Alkynyl sugar analogs for labeling and visualization of glycoconjugates in cells

ABSTRACT

Methods for metabolic oligosaccharide engineering that incorporates derivatized alkyne-bearing sugar analogs as “tags” into cellular glycoconjugates are disclosed. Alkynyl derivatized Fuc and alkynyl derivatized ManNAc sugars are incorporated into cellular glycoconjugates. Chemical probes comprising an azide group and a visual or fluorogenic probe and used to label alkyne-derivatized sugar-tagged glycoconjugates are disclosed. Chemical probes bind covalently to the alkynyl group by Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition and are visualized at the cell surface, intracellularly, or in a cellular extract. The labeled glycoconjugate is capable of detection by flow cytometry, SDS-PAGE, Western blot, ELISA, confocal microscopy, and mass spectrometry.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/079,226filed Mar. 24, 2008, titled “Alkynyl sugar analogs for the labeling andvisualization of glycoconjugates in cells,” and issued as U.S. Pat. No.7,960,139 on Jun. 14, 2011, which claims priority to U.S. ProvisionalPatent Application Ser. No. 60/896,777, filed on Mar. 23, 2007, titled“Pro-alkynyl sugar analogs for the labeling and visualization ofglycoconjugates in cells,” the contents of which are incorporated intheir entirety by reference as if fully disclosed herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support for research from theNational Institutes of Health and The Skaggs Institute for ChemicalBiology. The Government may have certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure provides a method for metabolic oligosaccharideengineering which uses azido and/or alkyne-bearing sugar analogs and/orprecursors of fucose and sialic acid to incorporate azido and/or alkynetags into cellular glycans that are fucosylated and sialylated. Thederivatized glycan is labeled by a chemical probe comprising an azidegroup and a visualizable, isolatable, and/or fluorogenic group. Thechemical probe binds covalently (labels) to alkynyl and/or azido groupsdisplayed in cellular glycans via copper (I)-catalyzed [3+2]azide-alkyne cycloaddition (CuAAC) or click chemistry. The labeledglycans can be visualized at the cell surface, intracellularly, or in acellular extract.

BACKGROUND OF THE INVENTION

Glycans are integral components of biological systems with far reachingactivities, many of which are only beginning to be understood. Glycansconstitute the most abundant and diverse class of biomolecules found innatural systems, consisting of oligosaccharide chains that are presentas independent polysaccharides (e.g., cellulose, an important structuralcomponent in plants; and heparin sulfate, an import factor of bloodclotting in mammals) or as glycoconjugates with lipids (glycolipids),proteins (glycoproteins, proteoglycans), and small molecule naturalproducts (e.g., antibiotics such as erythromycin, vancomycin, andteicoplanin).

Glycans play a role in almost every aspect of cellular activity. Mostglycans in higher eukaryotes are produced in the secretory pathway byglycosylation events, which entail the enzymatic transfer of saccharidesor oligosaccharide chains onto lipids and proteins. Proteinglycosylation is a complex co- or post-translational process thatmodifies the majority of the human proteome and serves a vast array ofbiological functions. Protein glycosylation exerts intrinsic effects onstructure, from mediating folding and oligimerization, to increasingstability, solubility, and circulation time. Inside of the cell, glycansaffect recognition, binding, targeting, and cellular distribution. Atthe cell surface, glycans are prominently displayed where they areinvolved in a host of molecular recognition events that modulateimportant physiological processes, such as cell-cell adhesion,inflammation, angiogenesis, coagulation, embryogenesis, differentiation,communication, and a myriad of other cellular signaling pathways.

Cell surface glycans have also been associated with physiologicaldysfunctions such as bacterial and viral infection, rheumatoidarthritis, and tumor progression. In the latter case, several types ofoncofetal and aberrant glycans have been established to correlate withmalignancy, invasiveness, inflammation and cancer metastasis. Inparticular, altered terminal fucosylation and sialylation, which arebelieved to result from changes in expression locations and levels offucosyltransferases (an group of enzymes that transfers a fucose from adonor substrate to an acceptor substrate, a glycoconjugate or glycan)and sialyltransferases (a group of enzymes that transfers a sialic acidfrom a donor substrate to an acceptor substrate, a glycoconjugate orglycan) respectively, are associated with tumor malignancy. For example,glycan determinants like Lewis y, Lewis x, sialyl Lewis x, sialyl Lewisa, sialyl Tn, Globo H, fucosyl GM1, and polysialic acid are expressed atelevated levels in neoplastic tissues. For this reason, these epitopesare promising and eagerly pursued targets for glycan-based vaccines.Additionally, several congenital glycosylation disorders, lysosomalstorage disorders, and immunological diseases have been linked withdysregulation of glycan catabolism/metabolism. Although known to beinvolved in physiological and pathophysiological events, theidentification of many glycan structures and delineation of their modeof action at the molecular level has been complicated by theirunderpinning complexity.

Glycan complexity results from many factors. They are synthesized in anon-templated, post-translational process, which means that sites ofglycoconjugate glycosylation and structures within them have proven,thus far, to be minimally predictable. This also means that glycanscannot be genetically manipulated in a similar fashion to DNA andproteins. Glycans are synthesized in the secretory pathway by a suite ofenzymes that are subject to multifaceted controls. The end glycanproducts can have enormous structural complexity (many possible glycanstructures, the diversity of which is also a function of the sugarbuilding blocks), structural micro-heterogeneity (multiple differentglycan structures attached to a glycoconjugate at the same position),and structural macro-heterogeneity (multiple sites and types of glycanattachment; for example, glycoproteins can be N-linked at Asn residues,or O-linked at Ser/Thr residues). Heterogeneity in glycan structuresappears to be dynamically regulated and functionally significant,governing multivalent interactions on the cell surface. Heterogeneityand multivalency complicate structure-function studies and the isolationof homogenous glycans in meaningful amounts from natural sources isnearly impossible. For the procurement of homogenousglycoconjugates/glycans synthesis is the only viable route, but remainsone of the most formidable challenges in glycobiology.

The link between glycan activity and complexity has presented majorchallenges to deciphering their activities on an individual protein, letalone, proteomic scale. Among the challenges facing global analysis aredevelopment of general methods for isolating glycans from complexproteomes; determining saccharide composition, site of proteinmodification, and fraction occupancy; and understanding the direct rolesof glycans in cellular function and dysfunction.

Specific glycan-tagging systems provide a powerful method for probingthe structure of heterogeneous glycans. The key to glycan taggingentails incorporating modified sugars derivatized with chemicalreporting groups into cellular glycans (typically via the normalbiosynthetic pathways, a process known as metabolic oligosaccharideengineering, or MOE) and then detecting the tagged-glycans by labelingtheir chemical reporting groups with a complementary probe thatchemically reacts with them in a specific manner (a chemoselectivemanner). Many selective chemical probing techniques have been used forprobing chemical reporting group-tagged glycoconjugates in cells. Thesemethods include bioorthogonal reactions such as ketoneaminooxy/hydrazideligation, Staudinger ligation, Michael addition, and thestrain-promoted, and Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition(CuAAC). Several chemical reporting groups are tolerated andsuccessfully incorporated into glycoconjugates using MOE, includingketones, thiols, photoreactive groups, azides, and alkynes. Thesereporting sugars have been labeled with tags such as FLAG peptides,biotin, and fluorescent or fluorogenic molecules. The strength of thesesystems is that the labeled glycan products have the potential to bemanipulated for specific glycan studies involving: enrichment andglycoproteomic analysis by means of mass spectrometry detection and/orquantitation by flow cytometry or visualization through microscopy toobtain information about glycan localization, trafficking, and dynamics.

The incorporation of exogenous natural or unnatural sugars into glycanshas been achieved by cellular biosynthetic pathways. These processesinvolve multistep enzymatic transformations that render free sugars inthe cytosol into nucleotide-donor sugars, the substrates forglycosyltransferases. In the case of fucose (Fuc), a salvage pathwayconsisting of Fuc kinase and GDP-Fuc (guanosine diphosphate fucose)pyrophosphorylase contributes to the production of GDP-Fuc, which isthen exploited by fucosyltransferases (FucTs) located in the Golgiapparatus to add Fuc onto glycoconjugates. Modifications at the6-position of Fuc are tolerated by the salvage pathway and FucTs. In thesialic acid (NeuAc) biosynthetic pathway, the precursorN-acetylmannosamine (ManNAc) is derived from GlcNAc or UDP-GlcNActhrough specific epimerases, then sequentially converted to sialic acid(NeuAc) by the cytosolic enzymes ManNAc 6-kinase, sialicacid-9-phosphate synthase, and sialic acid-9-phosphate phosphatase.CMP-NeuAc is subsequently formed in the nucleus, and transported to theGolgi apparatus for glycan elaboration by sialyltransferases. Studies onmetabolic delivery of N-acetyl mannosamine or ManNAc analogs show thatN-acyl chains up to five carbon atoms long are tolerated by the sialicacid biosynthetic pathway.

Prior glycoprotein probes have limited utility due to issues of cellulartoxicity. The incorporation of exogenous natural or unnatural sugarscomprising non-toxic probes into glycans by cellular biosyntheticpathways is important to study aberrant glycosylation. Furtherunderstanding of the molecular details and correlations between alteredglycosylation and pathological status is of great interest and is likelyto provide useful information for diagnosis and disease prognosis, inaddition to unveiling new therapeutic targets.

Glycosylation is the process of glycoconjugate synthesis and is animportant bioinformational process that occurs co- orposttranslationally on greater than 50% of eukaryotic proteins. Inliving organisms, it affects protein bioactivity and metabolic turnover.Inside of cells, it mediates protein folding, stability, andtrafficking. At the cell surface, glycans participate in molecularrecognition events that are central to biological and pathologicalprocesses like cell-cell interactions involved in adhesion, migration,and metastasis; host-pathogen interactions critical for bacterial andviral infections; and, initiation of immune response.

Aberrant glycosylation is often observed in pathological conditions suchas inflammation and cancer metastasis. In particular, altered terminalfucosylation and sialylation, which are believed to result from changesin expression locations and levels of fucosyltransferases andsialyltransferases, are associated with tumor malignancy. For example,glycan determinants like Lewis y, Lewis x, sialyl Lewis x, sialyl Lewisa, sialyl Tn, Globo H, fucosyl GM1, and polysialic acid are expressed atelevated levels in neoplastic tissues. For this reason, these epitopesare promising and eagerly pursued targets for glycan-based vaccines.However, cellular glycans are complex, heterogeneous populations,resulting from a non-template-driven process that cannot be manipulatedgenetically. This complexity makes the isolation and identification ofglycans for structural analysis one of the most challenging and definingtasks in glycobiology.

Specific glycan-tagging systems provide a powerful method for probingthe structure of heterogeneous glycans. The key to glycoconjugatetagging entails incorporating derivatized sugar chemical reportinggroups into cellular glycoconjugates (typically via the normalbiosynthetic pathways, a process known as metabolic oligosaccharideengineering, or MOE), and then detecting the tagged glycoconjugates bylabeling their chemical reporting groups with a complementary probe thatchemically reacts with them in a specific manner. Many selectivechemical probing techniques have been used for performing chemistry withchemical reporting group-tagged glycoconjugates in cells. These methodsinclude bioorthogonal reactions such as ketoneaminooxy/hydrazideligation, Staudinger ligation, Michael addition, and the strain-promotedand Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition.

Several chemical reporting groups are tolerated and successfullyincorporated into glycoconjugates using MOE, including ketones, thiols,photoreactive groups, azides, and alkynes. These reporting sugars havebeen labeled with tags, such as FLAG peptides, biotin, and fluorescentor fluorogenic molecules. The strength of these systems is that thelabeled glycan products have the potential to be manipulated forspecific glycan studies involving: enrichment and glycoproteomicanalysis by mass spectrometry; detection and/or quantitation by flowcytometry; or visualization through microscopy to obtain informationabout glycan localization, trafficking, and dynamics.

The incorporation of exogenous natural or unnatural sugars intoglycoconjugates is achieved by cellular biosynthetic pathways. Theseprocesses involve multistep enzymatic transformations that render freesugars in the cytosol into nucleotide-donor sugars, the substrates forglycosyltransferases. In the case of fucose (Fuc), a salvage pathwayconsisting of Fuc kinase and GDP-Fuc pyrophosphorylase contributes tothe production of GDP-Fuc, which is then exploited byfucosyltransferases (FucTs) located in the Golgi apparatus to add Fuconto glycoconjugates. Previous work has shown that modifications at the6-position of Fuc are tolerated by the salvage pathway and FucTs. In thesialic acid (NeuAc) biosynthetic pathway, the precursorN-acetylmannosamine (ManNAc) is derived from N-acetylglucosamine(GlcNAc) or uridine diphosphate GlcNAc (UDP-GlcNAc) through specificepimerases, then sequentially converted to sialic acid by the cytosolicenzymes ManNAc 6-kinase, sialic acid-9-phosphate synthase, and sialicacid-9-phosphate phosphatase. Cytosine monophosphate NeuAc (CMP-NeuAc)is subsequently formed in the nucleus, and transported to the Golgiapparatus for glycan elaboration by sialyltransferases. Studies onmetabolic delivery of ManNAc or its analogs show that N-acyl chains upto five carbon atoms long are tolerated by the sialic acid biosyntheticpathway.

Currently available glycoconjugate probes can be of limited utility dueto potential cellular toxicity. The incorporation of exogenous naturalor unnatural sugars comprising non-toxic probes into glycoconjugates bycellular biosynthetic pathways is important to study aberrantglycosylation which is often observed in pathological conditions such asinflammation and cancer metastasis. Further understanding of themolecular details and correlations between altered glycosylation andpathological status is of great interest and is likely to provide usefulinformation for diagnosis and disease prognosis, in addition tounveiling new therapeutic targets.

SUMMARY OF THE INVENTION

In one exemplary implementation, a method is disclosed comprisingpresenting an alkynyl-derivatized sugar to a cell, wherein thealkynyl-derivatized sugar has an alkynyl functional group, and whereinthe cell is capable of producing a glycoconjugate; incorporating thealkynyl-derivatized sugar into the cell, wherein the alkynyl-derivatizedsugar is subsequently used by the cell to produce a taggedglycoconjugate; wherein the tagged glycoconjugate includes: a glycanportion; a conjugate portion; and an alkynyl functional group; andreacting the tagged glycoconjugate with a probe to produce a labeled,tagged glycoconjugate.

In another exemplary implementation, the labeled glycoconjugate isdetected to determine the location of the labeled glycoconjugate in thecell.

In another exemplary implementation, the labeled glycoconjugate isdetected to determine the quantity of the labeled glycoconjugate in thecell.

In another exemplary implementation, the labeled glycoconjugate isdetected to determine the identity of the labeled glycoconjugate in thecell.

In another exemplary implementation, the alkynyl-derivatized sugar is analkynyl-derivatized fucose.

In another exemplary implementation, the alkynyl-derivatized sugar is analkynyl-derivatized fucose derivative.

In another exemplary implementation, the alkynyl-derivatized sugar is1,2,3,4-tetraacetyl alkynyl fucose or a 1,2,3,4-tetraacetyl alkynylfucose derivative.

In another exemplary implementation, the alkynyl-derivatized sugar is analkynyl-derivatized N-acetylmannosine or an alkynyl-derivatizedN-acetylmannosine derivative.

In another exemplary implementation, the alkynyl-derivatized sugar is asialic acid precursor.

In another exemplary implementation, the alkynyl-derivatized sugar is1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine.

In another exemplary implementation, the alkynyl-derivatized sugar isperacetylated.

In another exemplary implementation, the alkynyl-derivatized sugar isacetylated.

In another exemplary implementation, the alkynyl-derivatized sugar isManNAcyne.

In another exemplary implementation, the alkynyl-derivatized sugar isNeuAcyne.

In another exemplary implementation, the alkynyl-derivatized sugar isFucyne.

In another exemplary implementation, the alkynyl-derivatized sugar isbioorthogonal.

In another exemplary implementation, the alkynyl-derivatized sugar issubsequently incorporated into a glycoconjugate at a terminal position.

In another exemplary implementation, the alkynyl-derivatized sugar issubsequently incorporated into a glycoprotein.

In another exemplary implementation, the alkynyl-derivatized sugar issubsequently incorporated into a glycoprotein at a terminal position.

In another exemplary implementation, the alkynyl-derivatized sugar issubsequently incorporated into a glycolipid.

In another exemplary implementation, the alkynyl-derivatized sugar issubsequently incorporated into a glycolipid at a terminal position.

In another exemplary implementation, the alkynyl-derivatized sugar iscapable of fluorescence.

In another exemplary implementation, the alkynyl-tagged glycoconjugateis a fucosylated glycoconjugate.

In another exemplary implementation, the alkynyl-tagged glycoconjugateis a sialylated glycoconjugate.

In another exemplary implementation, the probe is azido-derivatized.

In another exemplary implementation, the probe reacts with thealkynyl-tagged glycoconjugate by azide-alkyne cycloaddition.

In another exemplary implementation, the azide-alkyne cycloadditionreaction is copper (I) catalyzed.

In another exemplary implementation, the probe-tagged glycoconjugatereaction generates a triazole moiety.

In another exemplary implementation, the triazole moiety is generatedwhile maintaining bioorthogonality of the functional groups.

In another exemplary implementation, the triazole moiety is generated atbiological pH.

In another exemplary implementation, the triazole moiety is generatedwith nearly 100% reaction efficiency.

In another exemplary implementation, the probe is fluorogenic andbecomes fluorescent upon azide-alkyne cycloaddition reaction with thetagged glycoconjugate.

In another exemplary implementation, the probe additionally comprises abiotin group.

In another exemplary implementation, the probe additionally comprises acoumarin group.

In another exemplary implementation, the coumarin probe is3-azido-7-hydroxycoumarin.

In another exemplary implementation, the detecting step comprisesvisualizing the location of labeled glycoconjugates by one or moretechniques of flow cytometry and confocal microscopy.

In another exemplary implementation, the detecting step comprisesquantifying the labeled glycoconjugates by one or more techniques offlow cytometry, SDS-PAGE, Western blot, ELISA, confocal microscopy, andmass spectroscopy.

In another exemplary implementation, the detecting step comprisesidentifying the labeled glycoconjugates by one or more techniques offlow cytometry, SDS-PAGE, Western blot, ELISA and confocal microscopy.

In another exemplary implementation, the incorporating step furthercomprises growing the cell in the presence of from about 1 to about 1000micromolar concentration of the alkynyl-derivatized fucose.

In another exemplary implementation, the incorporating step comprisesgrowing the cell in the presence of from about 50 to about 400micromolar concentration of the alkynyl-derivatized fucose.

In another exemplary implementation, the incorporating step comprisesgrowing the cell in the presence of from about 1 to about 100 micromolarconcentration of the alkynyl-derivatized N-acetylmannosamine.

In another exemplary implementation, the incorporating step comprisesgrowing the cell in the presence of from about 5 to about 50 micromolarconcentration of the alkynyl-derivatized N-acetylmannosamine.

In another exemplary implementation, the labeled glycoconjugate in thecell is on the surface of the cell.

In another exemplary implementation, the cells are permeabilized priorto labeling.

In another exemplary implementation, a method is disclosed comprisingpresenting an alkynyl-derivatized sugar to a cell, wherein thealkynyl-derivatized sugar has an alkynyl functional group, and whereinthe cell is capable of producing a glycoconjugate; incorporating thealkynyl-derivatized sugar into the cell, wherein the alkynyl-derivatizedsugar is subsequently used by the cell to produce a taggedglycoconjugate; wherein the tagged glycoconjugate includes a glycanportion; a conjugate portion; and an alkynyl functional group; andreacting the tagged glycoconjugate with a probe to produce a labeled,tagged glycoconjugate; wherein the resultant toxicity of the method isimproved by at least 10% as compared to presenting an azido-derivatizedsugar to produce the tagged glycoconjugate.

In another exemplary implementation, the resultant toxicity is improvedby at least 50%.

In another exemplary implementation, a compound is disclosed comprising:an alkynyl tagged glycoconjugate; and an azido-derivatized probe;wherein the alkynyl tagged glycoconjugate and azido-derivatized probeare joined via a triazole moiety.

In another exemplary implementation, the compound is fluorogenic.

In another exemplary implementation, the resultant toxicity measuredwhen the compound is presented to a cell or cells is increased by nomore than 10% as compared to the toxicity measured in a cell or cells towhich no compound is presented.

In another exemplary implementation, the azido-derivatized probe furthercomprises a biotin-labeled moiety.

In another exemplary implementation, the azido-derivatized probe furthercomprises an antibody-labeled moiety.

In another exemplary implementation, an alkynyl-derivatized fucose isformed by the process of: obtaining L-(+)-galactonic acid γ-lactone;transforming, L-(+)-galactonic acid γ-lactone to1,2:3,4-Di-O-isopropylidene-α-L-galactose by treatment with AmberliteIR120 and NaBH₄; transforming the hydroxyl group at position 6 of1,2:3,4-Di-O-isopropylidene-α-L-galactose to an alkynyl group bySeyferth-Gilbert homologation, or specifically first by treatment withPCC and NaOAc; filtering this mixture through a bed of silica gel, andthen treating the filtrate with a suspension of tBuOK and(EtO)₂P(O)CHN₂, thus creating6,7-deoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-ynopyranoside,referred to as 6-alkynylfucose diacetonide, or the diisopropylidene-Fucintermediate; removing the diacetonide protecting groups from6-alkynylfucose diacetonide to form 6-alkynyl fucose; and acetylatingthe resultant deprotected product 6-alkynyl fucose to form1,2,3,4-tetraacetyl alkynyl fucose, as a mixture of pyranoside andfuranoside forms.

In another exemplary implementation, an azido-derivatized fucose isprepared by the process of: obtaining 1-(+)-galactonic acid γ-lactone;transforming L(+)-galactonic acid γ-lactone to1,2:3,4-Di-O-isopropylidene-α-L-galactose by treatment with AmberliteIR120 and NaBH₄; transforming the hydroxyl group at position 6 of1,2:3,4-Di-O-isopropylidene-α-L-galactose to an azido group by treatmentwith TsCl and NaN₃ to create6,7-deoxy-1,2:3,4-di-O-isopropylidene-α-L-Fucose-6-azide, referred to as6-azidofucose diacetonide, or the diisopropylidene-Fuc intermediate;removing the diacetonide protecting groups from 6-alkynylfucosediacetonide to form 6-alkynyl fucose; and acetylating the resultantdeprotected product 6-alkynyl fucose to form 1,2,3,4-tetraacetyl alkynylfucose, as a mixture of pyranoside and furanoside forms.

In another exemplary implementation, an alkynyl-tagged glycoconjugate ismade by the process of fucosylating a glycoconjugate with the1,2,3,4-tetraacetyl alkynyl fucose by endogenous cellular metabolicpathways for glycan synthesis.

In another exemplary implementation, a compound is made by the steps offucosylating a glycoconjugate with the 1,2,3,4-tetraacetyl alkynylfucose by endogenous cellular metabolic pathways for glycan synthesis;and coupling the azido-derivatized probe with the fucosylatedglycoconjugate at least partially comprised of 1,2,3,4-tetraacetylalkynyl fucose via cycloaddition.

In another exemplary implementation, an alkynyl ManNAc-taggedglycoconjugate is made by the process of: obtaining D-mannosidehydrochloride; reacting the D-mannoside hydrochloride withN-succinimidyl 4-pentynoate to yield alkynyl ManNAc derivative;acetylating the alkynyl ManNAc derivative; and sialylating aglycoconjugate with the acetylated alkynyl ManAc derivative.

In another exemplary implementation, a fluorescent glycoconjugate ismade by the process of: obtaining D-mannoside hydrochloride; reactingthe D-mannoside hydrochloride with N-succinimidyl 4-pentynoate to yieldalkynyl ManNAc derivative; acetylating the alkynyl ManNAc derivative;sialylating a glycoconjugate with the acetylated alkynyl ManNAcderivative; and coupling an azido-derivatized probe with the sialylatedglycoconjugate at least partially comprised of the acetylated alkynylManNAc derivative via cycloaddition.

In another exemplary implementation, A method is disclosed comprisingthe steps of: presenting an alkynyl-derivatized sugar to a cell, whereinthe alkynyl-derivatized sugar has an alkynyl functional group, andwherein the cell is capable of producing a glycoconjugate; incorporatingthe alkynyl-derivatized sugar into the cell, wherein thealkynyl-derivatized sugar is subsequently used by the cell to produce atagged glycoconjugate; wherein the tagged glycoconjugate includes aglycan portion; a conjugate portion; and an alkynyl functional group;reacting the tagged glycoconjugate with a probe to produce a labeled,tagged glycoconjugate; detecting the labeled glycoconjugate; anddifferentially analyzing the proteomes of the cells incorporatingdetected, labeled glycoconjugate.

In another exemplary implementation, the cells are H. pylori or H.pylori-infected cells.

In another exemplary implementation, a method is disclosed comprisingthe steps of: providing an alkynyl-derivatized sugar to a cellpopulation, wherein the alkynyl-derivatized sugar has an alkynylfunctional group, and wherein the cells are capable of producing aglycoconjugate; incorporating the alkynyl-derivatized sugar into thecell, wherein the alkynyl-derivatized sugar is subsequently used by thecells to produce a tagged glycoconjugate, wherein the taggedglycoconjugate includes a glycan portion; a conjugate portion; and analkynyl functional group; reacting the tagged glycoconjugate with aprobe to produce a labeled, tagged glycoconjugate; visualizing thelabeled, tagged glycoconjugate population of the cells; anddifferentially analyzing the subset of cells expressing labeled, taggedLewis antigen epitopes.

In another exemplary implementation, a method is disclosed comprisinggenerating antibodies to the subset of cells expressing Lewis antigenepitopes.

In another exemplary implementation, cells are presented withderivatized sugars for a limited period of time.

In another exemplary implementation, the limited period of time is 30minutes.

In another exemplary implementation, derivatized sugars are presented toa cell for a limited time, and the presenting step is succeeded bypresenting the cell with non-derivatized sugars.

In another exemplary implementation, derivatized sugars are presented toa cell for a limited time, and both preceded and succeeded by presentingthe cell with non-derivatized sugars.

In another exemplary implementation, derivatized sugars are subsequentlylabeled and detected at various time intervals subsequent to the limitedpresentment of such sugars to the cell.

In another exemplary implementation, various time interval detections ofderivatized sugars are compared so as to assess cellular trafficking ofglycoconjugates.

In another exemplary implementation, differential cellular traffickingof glycoconjugates is assessed.

In another exemplary implementation, various time interval detections ofderivatized sugars are compared with various interval detections of thelocation of various intracellular and extracellular bodies (e.g.nucleus, Golgi apparatus, lysosome) so as to assess differentialcellular trafficking of glycoconjugates.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time are alkynyl-derivatized sugars.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time are azido-derivatized sugars.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time are both alkynyl andazido-derivatized sugars.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time are incorporated into fucosylatedglycoconjugates.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time are incorporated into sialylatedglycoconjugates.

In another exemplary implementation, derivatized sugars that arepresented to a cell for a limited time and are preceded and succeeded bypresenting the cell with non-derivatized sugars are incorporated intoboth fucosylated and sialylated glycoconjugates.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows analysis of cells labeled with Fuc analogs. FIG. 1(A) showsflow cytometry analysis of Jurkat cells treated with Fucalkynyl-derivatized analogs and labeled with biotin/fluoresceinconjugated streptavidin (filled trace, untreated cells; black, cellstreated with Fuc 3; grey, cells treated with alkynyl-derivatized Fuc 1).FIG. 1(B) shows dose-dependency of fucosyl-glycan tagging byalkynyl-derivatized Fuc 1 over 3 days. FIG. 1(C) shows the time courseof fucosyl glycan tagging by 200 micromolar alkynyl Fuc 1. FIG. 1 (D)shows cell growth analysis after treatment with different derivatizedsugar Fuc analogs: alkynyl Fuc 1, azido Fuc 2, control 3, and untreated.Jurkat cells were grown in the presence of 200 micromolar each Fucanalog for 3 days before cell numbers were counted. The data representthe percentage of treated cells vs. untreated cells (n=4).

FIG. 2 shows analysis of cell surface labeling of sialylglycoconjugates.

FIG. 2(A) shows flow cytometry analysis of Jurkat cells tagged withderivatized alkynyl-derivatized ManNAc (filled trace, untreated cells;green, cells treated with control 5; purple, cells treated with alkynylManNAc 4).

FIG. 2(B) shows dose dependency of sialyl glycoconjugate tagging withalkynyl-derivatized ManNAc 4 for 3 days.

FIG. 2(C) shows time course for tagging sialyl glycoconjugates bytreatment with 25 micromolar alkynyl-derivatized ManNAc 4.

FIG. 2(D) shows growth rate of Jurkat cells treated with different dosesof alkynyl-derivatized ManNAc 4 after 3 days.

FIG. 3 shows tagging of cell surface glycans by derivatized alkynylsugar analogs and subsequent labeling with probe3-azido-7-hydroxycoumarin 7. Shown is flow cytometry analysis of Jurkatcells tagged with 200 micromolar derivatized alkynyl Fuc 1 (A) or 25micromolar alkynyl-derivatized ManNAc 4 (B) for 3 days. The fluorescenceintensity was detected after labeling with a coumarin probe 7. Filledhistogram, cells treated with control sugar analog 3 or 5; openhistogram, cell treated with alkynyl-derivatized sugar 1 or 4.

FIG. 4 shows fluorescent imaging of fucosyl and sialyl glycoconjugatesin cells. Confocal microscopy of Hep3B cells treated with 200 micromolarFuc analogs or 25 micromolar ManNAc analogs. Cellular glycoconjugateswere biotin-labeled and stained with streptavidin (fluorescein, green),WGA lectin (Alexa Fluor 594, red), and Hoechst 33342 (blue). (Scalebars: 20 micrometer).

FIG. 5 shows visualization of derivatized alkynyl-tagged sialylglycoconjugates in cells using “click-activated” fluorogenic labeling.7. Shown is confocal microscopy of coumarin-labeled Hep3B cells. Cellswere treated with 25 micromolar derivatized ManNAc 5 or 4 for 3 days,and then labeled with fluorogenic coumarin probe 7 (blue) and stainedwith WGA lectin (Alexa Fluor 594, red). (Scale bars: 20 micrometer).

FIG. 6 shows detection of derivatized alkynyl-tagged glycoconjugates incell extracts subjected to Western blot. Glycoconjugates tagged withderivatized alkynyl sugars were labeled and subsequently detected byimmunoblotting of biotin tag (A) or fluorescent imaging of fluorogeniccoumarin probe 7 (B). Protein extracts from cells grown with differentsugars were analyzed (lane 1, control Fuc 3; lane 2, alkynyl-derivatizedFuc 1; lane 3, control ManNAc 5; lane 4, alkynyl-derivatized ManNAc 4).The protein gel (4-12%) was subsequently stained by Coomassie blue afterfluorescent imaging, to verify equal protein loading.

FIG. 7 shows ¹H-NMR spectra of peracetylated alkynyl Fuc 1.

FIG. 8 shows tagging of fucosyl glycans with derivatized alkynyl sugarson prostate cancer PC-3 and RWPE-1 prostate cells. Cells were treatedwith alkynyl-derivatized Fuc analog 1 or 3, labeled with azido-biotinprobe 6, and subjected to flow cytometry analysis. Filled histograms:cells treated with control Fuc 3; open histograms: cells treated withalkynyl-derivatized Fuc 1. Mean fluorescence intensity (MFI) of eachpeak is indicated.

FIG. 9 shows lectin staining of alkynyl-derivatized Fuc 1-tagged PC-3prostate cancer cells. Cells were treated with 200 micromolarderivatized alkynyl-derivatized Fuc 1, or Fuc 3, or left untreated.After three days, cells were labeled with biotin-conjugatedAAL/fluorescein-conjugated streptavidin (A) or fluorescein-conjugatedUEA-I (B). Fluorescent signal was detected by flow cytometry. AAL wasused to detect α-1,6- or α-1,3-linked Fuc; UEA-I was used to detectα-1,2-linked Fuc. Filled histogram: untreated cells without lectinstain; open histogram c: untreated cells stained with lectin; openhistogram b: control Fuc 3-treated cells stained with lectin; openhistogram a: alkynyl-derivatized Fuc 1-treated cells stained withlectin. AAL, Aleuria Aurantia Lectin; UEA-1, Ulex Europaeus AgglutininI.

FIG. 10 shows fluorescent imaging of fucosyl and sialyl glycoconjugatesin cells. Confocal microscopy of MCF-7 breast cancer cells treated with200 micromolar derivatized Fuc analogs (A) or 25 micromolar derivatizedManNAc analogs (B). Cells were biotin-labeled and stained withstreptavidin (fluorescein; green), WGA lectin (Alexa Fluor 594; red),and Hoechst 33342 (blue). Scale bars represent 20 micrometers.

FIG. 11 shows visualization of tagged sialyl glycoconjugates in cellsusing labeling via click-activated probe 7: confocal microscopy ofcoumarin-labeled MCF-7 cells. Cells were treated with 25 micromolarManNAc analogs 5 or 4, and then labeled with fluorogenic coumarin probe7 (blue) and stained with WGA lectin (Alexa Fluor 594; red). Scale barsrepresent 20 micrometers.

FIG. 12 shows detection of alkynyl-tagged glycoconjugates in cellextracts. Glycoconjugates tagged with alkynyl-derivatized sugars werelabeled and detected by azido rhodamine probe. Protein extracts fromcells grown with different sugars were analyzed by SDS-PAGE (12% gel),fluorescent imaging, and Coomassie Blue stain (lane 1: control Fuc 3;lane 2: alkynyl Fuc 1; lane 3: control ManNAc 5; lane 4: alkynyl ManNAc4).

FIG. 13 shows SDS-gel based derivatized fucosylated glycoproteomicprofiling of H. pylori. (A) Affinity purification of derivatizedalkynyl-tagged fucosylated proteins in various H. pylori strains afterlabeling with biotin probe. HS: gastric strain. HU: gastric ulcerstrain. HD: duodenal ulcer strain. HC: gastric cancer strain. (B)Protein identified from different strains of H. pylori by tagging withderivatized alkynyl Fuc and further labeling and subsequentvisualization and isolation of tagged glycoconjugates.

FIG. 14 shows examination of derivatized alkynyl-tagged fucosylatedglycoconjugates on CagA in H. pylori cancer strain byimmunoprecipitation and immunoblotting.

FIG. 15 shows time-lapse microscopy of pulse-chased Hep3b cells grownfor 30 minutes in medium containing Fucyne, with tagged glycoconjugatestructures subsequently labeled with biotin and detected usingstreptavidin-FITC as a secondary label at 2 hour intervals aftercompletion of the Fucyne pulse. Additional cellular structures arevisualized using alternative labeling systems (e.g., WGA lectin used tovisualize Golgi apparatus, and Hoechst used to visualize nucleus).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “abnormal” means an organism whose proteomediffers in identity (whether measure by individual or total proteinidentity), relative ratio, and/or glycosylation status of measurablecellular proteins.

As used herein, the term “alkynyl group” or “alkyne functional group”means an alkyne functional group (also called acetylene functionalgroup), which is a hydrocarbon comprised of a triple bond between twocarbon atoms.

As used herein, the term “alkynyl-derivatized sugar” means a syntheticsugar analog, in pro-molecular, metabolic precursor, and/or downstreammetabolite form, substituted with an alkynyl group.

As used herein, the term “alkynyl-derivatized” means a molecule in whichat least one carbon is substituted with an alkynyl functional group.

As used herein, the term “alkynyl functional group” means a chemicalmoiety consisting of at least one triple bond between two carbon atoms,with the formula C_(n)H_(2n-2).

As used herein, the term “alkynyl-tagged”, means a glycoconjugateincorporating an alkynyl-derivatized sugar.

As used herein, the terms “alkynyl fucose,” “alkynyl Fuc” and “Fucyne”are used interchangeably.

As used herein, the term “alkynyl N-acetylmannosamine,” “alkynyl ManNAc”and “ManNAcyne” are used interchangeably.

As used herein, the term “alkynyl sialic acid,” “alkynyl NeuAc” and“NeuAcyne” are used interchangeably.

As used herein, the term “antibody” means proteins that are found inblood or other bodily fluids of vertebrates, and are used by the immunesystem to identify and neutralize foreign objects, such as bacteria andviruses.

As used herein, the term “azido-derivatized” means a molecule in whichat least one carbon is substituted with an azido functional group.

Amino acid residues in peptides shall hereinafter be abbreviated asfollows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine isIle or I; Methionine is Met or M; Valine is Val or V; Serine is Ser orS; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A;Tyrosine is Tyr or Y; Histidine is H is or H; Glutamine is Gln or Q;Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D;Glutamic Acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W;Arginine is Arg or R; and Glycine is Gly or G. For further descriptionof amino acids, please refer to Proteins: Structure and MolecularProperties by Creighton, T. E., W. H. Freeman & Co., New York 1983.

As used herein, the term “bioorthogonal” means chemical reactants andreactions that are compatible with living systems. Bioorthogonalreactions proceed in high yield under physiological conditions andresult in covalent bonds between reactants that are otherwise stable inthese settings.

As used herein, the term “bioorthogonal chemical reporting group” meansa non-native, non-perturbing, inert chemical functional group, which canbe modified in biological systems by chemo-selective reactions withexogenously delivered probes.

As used herein, the term “binding moiety” means a moiety or functionalgroup capable of binding to a second chemical entity.

As used herein, the term “cellular glycan” or “cell glycan” refers to aglycan (either alone or as part of a glycoconjugate) that may be at thecell surface, intracellular, or within a cell lysate.

As used herein, the term “capable of producing” means that a cell isable to perform the designated biochemical function via a known orunknown biosynthetic pathway; for example, many cells are able toproduce glycosylated proteins through the FucT salvage pathway.

As used herein, the term “capturing” means chemically linking a moleculeof interest with a physical support, wherein the molecule of interest isimmobilized.

As used herein, the term “chemoselective” means the preferentialreaction of a chemical reagent with only one out of two or moredifferent available functional groups.

As used herein, the term “coumarin” means any of a group of fluorogeniccompounds related to benzopyrone or 2-chromenone that are capable offluorescence modulation dependent on position of substitution andidentity of functional groups.

As used herein, the term “conjugate portion” means a non-sugar portionof a glycoconjugate.

As used herein, the term “click-activated” means any reaction thatbioorthogonally proceeds in a manner that changes the chemical and/orphysical properties of the resultant molecule.

As used herein, the term “cycloaddition” means a chemical cyclizationreaction, in which two π bonds are lost and two σ bonds are gained—thereaction can proceed catalyzed or uncatalyzed or in a concerted orstepwise manner.

As used herein, the term “differential modification of +1 Da” means anamino acid that may bear a chemical modification resulting in amolecular weight shift of 1 dalton (Da). For example, a Asn residue witha N-linked bond to a glycan can be hydrolyzed to Asp, resulting in a +1Da change in molecular weight. A differential modification is added tosearching algorithms for MS peptide sequencing when all residues of aparticular amino acid are not modified (e.g., only Asn residues formerlycovalently bound to a glycan will have the +1 Da differentialmodification). Searching with a diff mod determines if and where a shiftfrom the Asn residue to an Asp residue has occurred, and thereforeassigns formerly N-glycosylated sites.

As used herein, the term “derivatization” is used to describe atechnique used in chemistry which transforms a chemical compound into aproduct of similar chemical structure, called a derivative. For example,when reference is made to a sugar analog or precursor that has been“derivatized” with an alkyne group, it is meant that the sugar analog isbearing an alkynyl group.

As used herein, the term “determining” means measuring (qualitatively orquantitatively) a chemical or physical characteristic of a sample ofinterest.

As used herein, the term “differential analysis” means assessment ofrelative quantities and identities of proteomes as compared amongheterogeneous samples or organisms.

As used herein, the term “epitope” means the part of a macromoleculethat is recognized by the immune system, specifically by antibodies, Bcells, or T cells.

As used herein, the term “flow cytometry” or “FACS” means a techniquefor examining the physical and chemical properties of particles or cellssuspended in a stream of fluid, through optical and electronic detectiondevices.

As used herein, the term “fluorescent labeled” means derivatizing amolecule with a fluorescent material.

As used herein, the term “fluorogenic” or “fluorescent reporting group”means a material capable of supporting a chemical reaction dependent onthe presence of a particular analyte material. Said analyte-dependentchemical reaction produces a fluorescent reporting molecule.

As used herein, the term “fluorescent” means a material exhibitingfluorescence.

As used herein, the term “fucose” is interchangeable with itsabbreviation (Fuc) and means a six-carbon deoxy pyran sugar,distinguished from other hexoses by a L-configuration and anunsubstituted carbon at the 6-position.

As used herein, the term “fucosyltransferase (FucT)” means an enzymethat transfers a fucose from a donor substrate, GDP-fucose(GDP=Guanosine diphosphate), to an acceptor substrate, a glycoconjugateor glycan.

As used herein, the term “fucosylated” means a molecule (typically aglycoconjugate or glycan) that has been covalently appended with a Fucresidue (typically by a FucT).

As used herein, the term “functional group” (or “moiety”) means aspecific group of atoms within molecules that are responsible for thecharacteristic chemical reactions of those molecules. The samefunctional group will undergo the same or similar chemical reaction(s)regardless of the size of the molecule it is a part of. However, itsrelative reactivity can be modified by nearby functional groups.

As used herein, the term “GDP analog” means a molecular derivative ofGuanosine diphosphate (GDP).

As used herein, the term “glycan” refers to a polysaccharide, oroligosaccharide. Glycan is also used herein to refer to the carbohydrateportion of a glycoconjugate, such as a glycoprotein, glycolipid,glycopeptide, glycoproteome, peptidoglycan, lipopolysaccharide or aproteoglycan. Glycans are typically comprised of monosaccharides linkedtogether with O-glycosidic bonds. For example, cellulose is a glycan (ormore specifically a glucan) composed of β-1,4-linked D-glucose, andchitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamineGlycans can be homo or heteropolymers of monosaccharide residues, andcan be linear or branched. Glycans can be found attached to lipids andproteins, as in glycoproteins and proteoglycans. They are generallyfound on the exterior surface of cells. O- and N-linked glycans are verycommon in eukaryotes but may also be found, although less commonly, inprokaryotes. N-linked glycans are attached through amide bonds toasparagine residues found in the N-glycosylation consensus sequon. Thesequon is a Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acidexcept proline. O-linked glycans are attached through glycosidic bondswith oxygen groups on serine and threonine residues in proteins, orhydroxyl groups of lipids and small molecules.

As used herein, the term “glycoconjugate” means a molecule covalentlymodified with glycans.

As used herein, the term “glycoprotein” means a protein covalentlymodified with glycan(s). There are four types of glycoproteins: 1)N-linked glycoproteins, 2) O-linked glycoproteins (mucins), 3)glucosaminoglycans (GAGs, which are also commonly called proteoglycans),4) GPI-anchored. Most glycoproteins have structural micro-heterogeneity(multiple different glycan structures attached within the sameglycosylation site), and structural macro-heterogeneity (multiple sitesand types of glycan attachment).

As used herein, the term “glycoproteomics” refers to a branch ofproteomics that identifies, catalogs, and characterizes proteinscontaining carbohydrates as a post-translational modification.Glycoproteomics also refers to the study of a cell, tissue, ororganism's glycan and glycoprotein content at any point in time.

As used herein, the term “glycosylation” means the enzymatic transfer ofsaccharides or oligosaccharide chains onto glycoconjugates. Proteinglycosylation is a complex co- or post-translational process thatmodifies the majority of the human proteome, vastly expanding itsfunctional repertoire.

As used herein, the term “harvesting” means concentrating, collecting,purifying and/or storing a material of interest.

As used herein, the term “isolated” means glycoconjugates that can beselectively separated by secondary detection means.

As used herein, the term “incorporating” means introducing a compound orderivative of a compound into the intracellular environment by anymethod, including but not limited to inclusion in media or restrictedmedia; electroporation; injection; phagocytosis; active transport;endocytosis; active transport; passive transport; carrier-assistedtransport; vesicle-mediated transport; and diffusion.

As used herein, the term “labeled glycoprotein” means a glycoproteincovalently attached via cycloaddition to a moiety that can facilitatethe manipulation of the “labeled glycoprotein,” such as the isolation,visualization, detection, and quantification of the labeledglycoprotein.

As used herein, the term “liquid chromatography-mass spectrometry” or“LC-MS” refers to an analytical chemistry technique that combines thephysical separation capabilities of liquid chromatography (aka HPLC)with the mass analysis capabilities of mass spectrometry (MS). LC-MS isa powerful technique used for many applications which has very highsensitivity and specificity. Generally its application is orientedtowards the specific detection and potential identification of chemicalsin the presence of other chemicals (in a complex mixture). LC-MS is alsoused in the study of proteomics where components of a complex mixturemust be detected and identified in some manner. The bottom-up proteomicsLC-MS approach to proteomics generally involves protease digestion(usually Trypsin) followed by LC-MS with peptide mass fingerprinting orLC-MS² (tandem MS) to derive the sequence of individual peptides.

As used herein, the term “metabolic oligosaccharide engineering” or“MOE” means the process of incorporating an alkynyl-derivatized sugarinto a glycoconjugate.

As used herein, the term “MudPIT” or Multidimentional ProteinIdentification Technology refers to the characterization of proteinmixtures using tandem LC-MS². A peptide mixture that results fromdigestion of a protein mixture is fractionated by multiple steps ofliquid chromatography. The eluent from the chromatography stage can beeither directly introduced to the tandem MS through electrosprayionization, or laid down on a series of small spots for later massanalysis using MALDI.

As used herein, the term “proteome” refers to the entire complement ofproteins expressed by a genome, cell, tissue or organism. Morespecifically, it is the expressed proteins at a given time point underdefined conditions.

As used herein, the term “presenting” means introducing into theextracellular environment, including, but not limited to, inclusion ingrowth media, restricted media, reaction solution, buffer, and/orstaining solution.

As used herein, the term “proteomics” refers to the study of theproteome. Proteomics has largely been practiced through the separationof proteins by two dimensional gel electrophoresis. In the firstdimension, the proteins are separated by isoelectric focusing, whichresolves proteins on the basis of charge. In the second dimension,proteins are separated by molecular weight using SDS-PAGE. The gel isdyed with Coomassie Blue or silver stain to visualize the proteins.Spots on the gel are proteins that have migrated to specific locations.The mass spectrometer has augmented proteomics. Peptide massfingerprinting identifies a protein by cleaving it into short peptidesand then deduces the protein's identity by matching the observed peptidemasses against a sequence database. Tandem mass spectrometry, on theother hand, can get sequence information from individual peptides byisolating them, colliding them with a non-reactive gas, and thencataloging the fragment ions produced.

As used herein, the term “pulse-chase” means a method for examining acellular process occurring over time by successively exposing the cellsto a labeled compound (pulse) and then to the same compound innonlabeled form (chase).

As used herein, the term “reacting” means inducing a chemical reactionbetween two or more substances, including, but not limited to,catalyzing such reaction and providing appropriate supporting reactionsubstituents to maintain biochemical pH and thermodynamic environments.

As used herein, the term “reporting group” means a molecule that hasproperties capable of presenting detectable feedback about eventstranspiring in a test system (from a controlled in vitro assay to acomplex biological system).

As used herein, the term “sialylated” means a molecule (typically aglycoconjugate or glycan) that has been covalently appended with asialic acid (NeuAc) residue (typically by a sialyl transferase)

As used herein, the term “tagged” means a glycoconjugate that hasincorporated an alkynyl-derivatized sugar through any permissivebiosynthetic pathway involved in glycoconjugate synthesis.

As used herein, the term “toxicity” means the relative percentage ofcells (assayed by any method of cell counting) surviving 3 days in vitroor in vivo after addition of sugar analogs (natural and/or derivatized)compound to the relevant cellular environment.

As used herein, the term “trafficking” means the movement of materialfrom one location to another within, into, or out of a cell, and anyassociated modifications of the material occurring in the process.

In one exemplary implementation, the disclosure provides a method oflabeling glycoconjugates in a cell, the method comprising: presenting analkynyl-derivatized sugar; incorporating the alkynyl-derivatized sugarinto glycoconjugates in the cell by growing the cell in the presence ofthe alkynyl-derivatized sugar to create an tagged glycoconjugate(alkynyl-tagged glycoconjugate); contacting the tagged glycoconjugatewith a chemical probe wherein said chemical probe reacts with saidalkynyl group in the tagged glycoconjugate to create a labeled, taggedglycoconjugate; and manipulating the labeled, tagged glycoconjugate forfurther analysis. Analysis can include detecting labeledalkynyl-derivatized-tagged glycoconjugates through fluorescence todetermine one or more of the location and relative abundance; orisolating them to determine their identity and relative abundance.

In one exemplary implementation, the disclosure provides a method oflabeling fucosylated glycoconjugates in a cell, the method comprising:presenting an alkynyl-derivatized fucose; tagging a glycoconjugate inthe cell by growing the cell in the presence of an alkynyl-derivatizedfucose to create an alkynyl-tagged fucosylated glycoconjugate; labelingthe alkynyl-tagged glycoconjugate with a chemical probe which will bindcovalently to the alkynyl group to create a labeled-glycoconjugate; anddetecting the labeled, tagged glycoconjugate to determine that thelabeled-glycoconjugate in the cell is a fucosylated glycoconjugate.

In another exemplary implementation, the disclosure provides a method ofidentifying a sialylated glycoconjugate in a cell, the methodcomprising: presenting an alkynyl-derivatized N-acetylmannosamine;tagging a glycoconjugate in the cell by growing the cell in the presenceof alkynyl-derivatized N-acetylmannosamine to create a tagged,sialylated alkynyl-derivatized glycoconjugate; labeling thealkynyl-derivatized glycoconjugate with a chemical probe which will bindcovalently to the alkynyl group to create a labeled, taggedglycoconjugate; and detecting the labeled-glycoconjugate to determinethat the labeled-glycoconjugate in the cell is a sialylatedglycoconjugate.

In a further exemplary implementation, the disclosure provides a methodof incorporating an alkynyl derivatized sugar into a glycoconjugate in acell, the method comprising: presenting an alkynyl-derivatized sugar;and tagging a glycoconjugate in the cell by growing the cell in thepresence of the alkynyl-derivatized sugar to create an labeled, taggedglycoconjugate.

In one exemplary implementation, the alkynyl-derivatized sugar taggedglycoconjugate is a fucosylated glycoconjugate and thealkynyl-derivatized sugar is an alkynyl-derivatized fucose. In aspecific exemplary implementation, the alkynyl-derivatized fucose is1,2,3,4-tetraacetyl alkynyl fucose.

In another exemplary implementation, the tagged glycoconjugate is asialylated-glycoconjugate and the alkynyl-derivatized sugar is analkynyl-derivatized N-acetylmannosamine. In a specific exemplaryimplementation, the alkynyl-derivatized N-acetylmannosamine is1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine.

In another exemplary implementation, the disclosure provides a method ofdetecting an alkynyl-tagged glycoconjugate, the method comprising:contacting the alkynyl-derivatized sugar tagged glycoconjugate with achemical probe wherein said chemical probe reacts with said alkynylgroup in the alkynyl-derivatized sugar-tagged glycoconjugate to create alabeled, tagged glycoconjugate; and detecting the labeled, taggedglycoconjugate to determine one or more of the location and theabundance of the labeled-glycoconjugate in the cell. In one exemplaryimplementation, the contacting step is performed on a cell surface, on apermeabilized cell, or on a cellular extract.

In one exemplary implementation, the disclosure provides a method ofmetabolic oligosaccharide engineering (MOE) that incorporatesderivatized alkyne-bearing sugar analogs into cellular glycoconjugates,thereby creating alkynyl-tagged glycoconjugates.

In one exemplary implementation, the alkyne-derivatized sugar analogsutilized in MOE are minimally toxic to the cell.

In one exemplary implementation, the alkyne-derivatized sugar analogsutilized in MOE minimally alter the cell's normal proteosomeglycosylation pattern.

In one exemplary implementation, the derivatized alkynyl sugars areperacetylated.

In one exemplary implementation, the derivatized alkynyl sugars areacetylated.

In one exemplary implementation, the derivatized alkynyl sugars arederivatized fucose (Fuc).

In one exemplary implementation, the derivatized alkynyl sugars arefucose analog precursors capable of subsequent intracellularderivatization and subsequent incorporation into cellular, cell surfaceand/or extracellular fucosylated glycoconjugates.

In one exemplary implementation, the derivatized alkynyl sugars aresialic acid precursors capable of subsequent derivatization andincorporation into cellular, cell surface and/or extracellularsialylated glycoconjugates.

In one exemplary implementation, the derivatized alkynyl sugars areManNAcyne.

In one exemplary implementation, the derivatized alkynyl sugars areNeuACyne.

In one exemplary implementation, the derivatized alkynyl sugars areFucyne.

In one exemplary implementation, the derivatized alkynyl sugars aremetabolic precursors to derivatized fucose and sialic acid analoguescapable of subsequent intracellular metabolic incorporation intofucosylated and/or sialylated glycoconjugates.

In one exemplary implementation, the derivatized alkynyl sugars arecapable of metabolic incorporation into fucosylated and/or sialylatedglycoconjugates where they are subsequently capable of azido-alkynylcycloaddition covalent binding with an azido-derivatized probe so as tocreate a labeled, tagged glycoconjugate.

In one exemplary implementation, the derivatized alkynyl sugars arebioorthogonal.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into glycoconjugates.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into glycoconjugates at the terminal position.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into fucosylated glycoconjugates.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into sialylated glycoconjugates.

In one exemplary implementation, derivatized alkynyl sugars capable offluorescence by further derivatization are incorporated intoglycoconjugates.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into glycoproteins.

In one exemplary implementation, derivatized alkynyl sugars areincorporated into glycolipids.

In one exemplary implementation, the glycoconjugate is a fucosylatedglycoconjugate or a sialylated glycoconjugate.

In another exemplary implementation, the glycoconjugate is a fucosylatedglycoconjugate and the alkynyl-derivatized sugar originates from analkynyl-derivatized fucose in the cell by MOE. In a specific exemplaryimplementation, the alkynyl-derivatized fucose is 1,2,3,4-tetraacetylalkynyl fucose.

In one exemplary implementation, the alkynyl-tagged glycoconjugate is asialylated-glycoconjugate and the alkynyl-derivatized sugar originatesfrom alkynyl-derivatized N-acetylmannosamine in the cell by MOE.

In a specific exemplary implementation, the alkynyl-derivatizedN-acetylmannosamine is 1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine.

In another exemplary implementation, the MOE sugar incorporating stepfurther comprises growing the cell in the presence of thealkynyl-derivatized fucose, from about 1 to about 1000 micromolarconcentrations in the growth medium.

In another exemplary implementation, the MOE sugar incorporating stepcomprises growing the cell in the presence of the alkynyl-derivatizedN-acetylmannosamine, from about 1 to about 100 micromolar concentrationin the growth medium.

In one exemplary implementation, the labeled-glycoconjugate is acellular glycoconjugate located on the surface of the cell. In anotherexemplary implementation, the method further comprises treating the cellto permeabilize the cell prior to the contacting step.

In another exemplary implementation, azide bearing probes additionallycomprising one or more of biotin and coumarin groups are boundcovalently to alkynyl-tagged glycoconjugates to provide labeledglycoconjugates.

In one exemplary implementation, tagged glycoconjugates are capable ofsubsequent chemoselective labeling.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition.

In one exemplary implementation, the probe is fluorogenic.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by CuAAC.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface in aqueoussolutions.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface at biologicallyrelevant pH.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface while maintainingbioorthogonality of the reaction components and products.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface at biological pH.

In one exemplary implementation, tagged glycoconjugates are labeled witha probe by azide-alkyne cycloaddition so as to generate a triazolemoiety at the tagged glycoconjugate-probe interface with nearlyquantitiative reaction efficiency.

In one exemplary implementation, the probe is an azido-derivatizedprobe.

In one exemplary implementation, the probe is a coumarin.

In one exemplary implementation, the probe is biotin.

In one exemplary implementation, the probe additionally includes ansecondary binding label.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified directly.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified indirectly through use of secondarybinding/detection means.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified through use of antibody-antigeninteractions.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified through use of lectin-glycan interactions.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified through use of streptavidin/avidin-biotinbinding.

In one exemplary implementation, the probe is additionally capable ofbeing isolated or quantified through use of a fluorophore.

In a further exemplary implementation, a variety of techniques aredisclosed for visualization of the labeled cellular glycoconjugate.

In one exemplary implementation, labeled, tagged glycoconjugates arevisualized on the cell surface of a eukaryotic or prokaryotic cell.

In one exemplary implementation, labeled, tagged glycoconjugates areisolated.

In one exemplary implementation, labeled, tagged glycoconjugates arevisualized.

In one exemplary implementation, labeled, tagged glycoconjugates areisolated through streptavidin/avidin-biotin interactions.

In one exemplary implementation, labeled, tagged glycoconjugates areisolated through antibody-antigen interactions.

In one exemplary implementation, labeled, tagged glycoconjugates arevisualized through azide-alkyne cycloaddition-mediated fluorescence.

In one exemplary implementation, labeled, tagged glycoconjugates arequantified through azide-alkyne cycloaddition-mediated fluorescence.

In one exemplary implementation, the chemical probe comprises an azidegroup. In a specific exemplary implementation, the chemical probe bindscovalently to the alkynyl group in tagged-glycoconjugates by CuAAC,thereby creating labeled-glycans.

In one exemplary implementation, the chemical probe further comprisesone of a visualizable probe and a fluorogenic probe. In one exemplaryimplementation, the visualizable probe comprises a biotin group. Inanother exemplary implementation, the fluorogenic probe comprises acoumarin group.

In one exemplary implementation, the detecting step comprisesvisualizing the labeled glycoconjugate by one or more techniques of flowcytometry, SDS-PAGE, Western blot, ELISA, confocal microscopy, and massspectrometry. In another exemplary implementation, the detecting stepfurther comprises quantifying the labeled-glycoconjugate by one or moretechniques of flow cytometry, SDS-PAGE, Western blot, ELISA and confocalmicroscopy.

In one exemplary implementation, derivatized sugars are presented to acell for a limited time, and succeeded by presenting the cell withnon-derivatized sugars.

In one exemplary implementation, derivatized sugars are presented to acell for a limited time, and both preceded and succeeded by presentingthe cell with non-derivatized sugars.

In one exemplary implementation, the derivatized sugars are subsequentlylabeled and detected at various time intervals subsequent to the limitedpresentment of such sugars to the cell.

In one exemplary implementation, the various time interval detections ofderivatized sugars are compared so as to assess cellular trafficking ofglycoconjugates.

In one exemplary implementation, the various time interval detections ofderivatized sugars are compared so as to assess differential cellulartrafficking of glycoconjugates.

In one exemplary implementation, the various time interval detections ofderivatized sugars are compared with various interval detections of thelocation of various intracellular and extracellular bodies (e.g.nucleus, Golgi apparatus, lysosome) so as to assess differentialcellular trafficking of glycoconjugates.

In one exemplary implementation, derivatized sugars are presented to acell for a limited time are alkynyl-derivatized sugars.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time are alkynyl-derivatized sugars.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time are azido-derivatized sugars.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time are both alkynyl and azido-derivatized sugars.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time are incorporated into fucosylatedglycoconjugates.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time, preceded and succeeded by presenting the cellwith non-derivatized sugars are incorporated into sialylatedglycoconjugates.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time, preceded and succeeded by presenting the cellwith non-derivatized sugars are incorporated into both fucosylated andsialylated glycoconjugates.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time, preceded and succeeded by presenting the cellwith non-derivatized sugars are presented to the cells with CuAACcatalysts.

In one exemplary implementation, the derivatized sugars presented to acell for a limited time, preceded and succeeded by presenting the cellwith non-derivatized sugars are presented to the cells without CuAACcatalysts.

Herein disclosed, alkynyl Fuc and alkynyl ManNAc analogs are synthesizedand utilized as reporting saccharides in a method to tag fucosylated andsialylated glycoconjugates in mammalian cells. Previously, a fluorescentlabeling technique for probing metabolically labeled fucosylatedglycoconjugates in cells was reported by this laboratory.

In the previous approach, derivatized azido Fuc analogs incorporatedinto glycoconjugates were labeled with a 1,8-naphthalimide fluorogenicprobe, by using Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition, oralkyne-azide “click” reaction. The click-activated fluorescent labelingwas used for specifically utilizing azido-derivatized labels to labelalkynyl-derivatized, sugar-tagged fucosylated and sialylatedglycoconjugates, and is also effective for use in so-called“pulse-chase” applications where there is limited presentment of theazido-derivatized sugar to the cell, and/or the presentment of theazido-derivatized sugar to the cell is in low concentration. In thepresent method, alkynyl Fuc and alkynyl ManNAc analogs show reducedtoxicity to cells when compared with azido Fuc analogs. Moreover, whenthese alkynyl derivatized sugars are coupled with biotin,click-activated fluorogenic coumarin, and other fluorescent probes, thismethod allows for the isolation of fucosylated and sialylatedglycoconjugates for further analysis, and fluorescent imaging (wherealkynyl sugar labeling causes less background signal). This method canbe used for visualizing glycan dynamics inside of cells and to identifyimportant glycan markers.

Synthesis of Alkynyl Sugars and Biotinylated Azide Probe.

In one exemplary implementation of the disclosure, an Intermediary ofAlkynyl Derivatized Fucose is 1,2:3,4-Di-O-isopropylidene-α-L-galactose,17. To L-galactono-1,4-lactone (10 g, 56.1 mmol) in MeOH (60 mL) andwater (250 mL) at 0° C. was added Amberlite IR 120 (H⁺) resin (50 mL).NaBH₄ (2.2 g, 56.1 mmol) was added portionwise, and the reaction mixturewas stirred for 1 h at room temperature. The resin was removed byfiltration, and the filtrate was evaporated. The residue was dissolvedin acetone (220 mL), CuSO₄ (22.2 g, 0.14 mol) and H₂SO₄ (1 mL) was addedand the solution was stirred at room temperature overnight. The CuSO₄was removed by filtration, and the filtrate was neutralized withCa(OH)₂. After removal of Ca(OH)₂ and concentration, the residue waspurified by flash column chromatography on silica gel (AcOEt/hexane 1:1)to afford 17 (9.1 g, 62%).

In one exemplary implementation of the disclosure, peracetylated alkynylderivatives of Fuc 1 and ManNAc 4, shown in Scheme 1, are synthesizedand used to tag fucosylated and sialylated glycoconjugates,respectively, in vivo. The sugar derivatives are synthesized in theirperacetylated forms, as this modification is known to increase theircellular uptake efficiency. The acetate esters are subsequentlyhydrolyzed in the cytosol.

In one aspect of the disclosure, the synthesis of alkynyl Fuc (1, seeExample 1, Scheme 2) proceeds from a known four-step transformation,beginning with L(+)-galactonic acid γ-lactone and ending with thealkynyl diisopropylidene-Fuc intermediate. Subsequent protecting groupremoval followed by acetylation of the intermediate yields the desiredcompound 1, as a mixture of pyranoside and furanoside forms. Thismixture is used directly for labeling fucosylated glycoconjugates incells.

In another aspect of the disclosure, compound 4 is synthesized and usedfor tagging sialylated glycoconjugates. D-Mannosamine hydrochloride isreacted with N-succinimidyl 4-pentynoate in triethylamine to yieldalkynyl ManNAc derivative (see Example 2, Scheme 3). The alkynyl ManNAc4 is subsequently obtained by acetylation.

The coupling partner, biotinylated azido probe 6, is synthesized bycoupling of biotin to 1-azido-3-aminopropane (see Example 4, Scheme 4).Fluorogenic probe 7, 3-azido-7-hydroxycoumarin, is synthesized asreported. Modified sugar analogs and probes used in this study areillustrated in Scheme 1.

Fluorescent Labeling of Alkynyl Glycoconjugates at the Cell Surface.

In another exemplary implementation, a method for labeling fucosylatedglycoconjugates at the cell surface is disclosed. In one aspect, Jurkatcells are grown in the presence of derivatized alkynyl Fuc 1. Aftertreatment, cells are subjected to CuAAC (click chemistry) to couplebiotinylated azido probe 6 with any alkynyl Fuc-bearing glycoconjugates,and stained with fluorescein-conjugated streptavidin.

Labeling alkynyl Fuc-bearing cell surface glycoconjugates is illustratedin FIG. 1. FIG. 1 shows analysis of cells tagged with Fuc analogsanalyzed by monitoring fluorescence intensity with flow cytometry afterlabeling with a biotin azide probe 6 and staining cells withfluorescein-conjugated streptavidin. As shown in FIG. 1A, thederivatized alkynyl Fuc 1-treated cells show increased fluorescenceintensity compared with control Fuc 3-treated cells, as analyzed by flowcytometry. This indicates that alkynyl-derivatized Fuc residues areincorporated into (“tag”) cell surface glycoconjugates and that thesetags can serve as binding sites for chemoselective cycloadditionlabeling. Without being bound by theory, incorporation of thederivatized Fuc analogs into fucosylated glycoconjugates likely occursvia the Fuc salvage pathway. Alkynyl Fuc analog 1-treated cells showed adose dependent increase of fluorescence signal, with a 3-fold greatermean fluorescence intensity (MFI) compared with Fuc treated cells at 200micromolar concentration (FIG. 1B). The data also showed saturation ofalkynyl Fuc 1 incorporation within one-day of incubation, although therewas a slight increase of labeling signal on cells treated for three dayswith alkynyl Fuc 1 (FIG. 1C).

It is also disclosed how treatment with exogenous Fuc analog affectscell growth rate. As shown in FIG. 1D, the number of cells after 3 daysis similar whether they are treated with 200 micromolar alkynyl Fuc 1,200 micromolar Fuc 3, or grown without exogenous Fuc. In contrast, theaddition of 200 micromolar azido Fuc 2 inhibits cell growthconsiderably, by 65% when compared with the untreated cells. Theseresults indicate that azido Fuc 2 analog, which was used previously forprobing fucosylation, is more toxic to cells than alkynyl Fuc 1 analog.Such toxicity may lead to global change in expression, therefore anontoxic probe is preferable for accurate probing of glycoconjugateexpression.

Previously, it was demonstrated that the majority of an exogenous ManNAcanalog, N-levulinoylmannosamine, acquired by cells is converted intosialic acid via biosynthetic pathways.

It is now disclosed in one exemplary implementation of the disclosurethat treating cells with derivatized alkynyl ManNAc 4 results inderivatized alkyne-bearing sialyl glycoconjugates. In one aspect of themethod, cells are treated with 4 at various concentrations for one to 3days. Modified sugar analogs and probes used in this disclosure areshown in Scheme 1. Tagging of the cell surface glycoconjugates byderivatized alkynyl ManNAc is illustrated in FIG. 2. FIG. 2 showsanalysis of cells labeled with ManNAc analogs analyzed by monitoringfluorescence intensity with flow cytometry after clicking on the biotinazide probe 6 and staining cells with fluorescein-conjugatedstreptavidin. Labeling with derivatized alkynyl ManNAc 4 yielded aspecific signal on the cell surface compared with the control valuesobtained from cells treated with control ManNAc 5 (FIG. 2A).Dose-dependent labeling was observed in cells treated with derivatizedalkynyl ManNAc 4 (FIG. 2B). Compared with the MFI of controls, there wassignificant labeling on cells treated with derivatized alkynyl ManNAc 4,even at concentrations as low as 3 micromolar (15-fold increase).Time-course experiments revealed that treatment with derivatized alkynylManNAc 4 from one to three days gave a 15- to 23-fold increase inlabeling intensity over control levels (FIG. 2C). The optimalconcentration of 4 for tagging sialyl glycoconjugates falls between 25and 50 micromolar. In this concentration range, 4 showed little or notoxicity, although it is more toxic above 100 micromolar (FIG. 2D).

One of the advantages of labeling an alkynyl-derivatized sugar taggedglycoconjugate with an azido-derivatized probe via CuAAC, or the clickreaction, is the formation of a triazole unit, which can modulate thefluorescent emission of probes through electron-donating properties. Itwas previously shown that such click-activated fluorescence is useful influorescently labeling azido Fuc-bearing glycoconjugates using a1,8-naphthalimide-alkyne probe. However, the azido version of thenaphthalimide probe causes high background, making it less useful forlabeling our alkynyl sugars.

Recently, another click-activated azido-deritatized fluorescent probe,based on coumarin, was reported in the literature. In one aspect of thedisclosure, the fluorogenic probe, 3-azido-7-hydroxycoumarin 7, is usedas the coupling partner for alkynyl tags on labeled glycoconjugates. Asshown in FIG. 3, cells treated with derivatized alkynyl Fuc 1 (FIG. 3A)or derivatized alkynyl ManNAc 4 (FIG. 3B) allowed significantfluorescent labeling after reacting with a 3-azido-7-hydroxycoumarinprobe, whereas cells treated with control sugars 3 and 5 gave very lowbackground signals, evidencing low reactivity with a3-azido-7-hydroxycoumarin probe.

Visualization of Fluorescently Labeled Glycoconjugates in Cells.

One exemplary implementation of the disclosure provides a method tovisualize the localization of glycoconjugates using alkynyl sugartagging. To visualize the localization of glycoconjugates tagged withalkynyl sugars, adherent cells are grown on slides in the presence orabsence of derivatized alkynyl sugars. After a 3-day-incubation, cellsattached to the slides are fixed, permeabilized, and labeled with eitherbiotin probe 6 or fluorogenic probe 7 for fluorescent signal analysiswith confocal microscopy (FIGS. 4, 5, 10 and 11). For comparison,samples are also stained with wheat germ agglutinin (WGA, a Golgimarker) and Hoechst 33342 (marker for cell nuclei). In one aspect of themethod, cancer cell lines, such as Hep3B (hepatocellular carcinoma) andMCF-7 (breast adenocarcinoma) cells, are treated with derivatizedalkynyl Fuc 1 to result in a strong punctate-labeling signal afterlabeling tagged glycoconjugates with a biotin probe 6 and staining withfluorescein-conjugated streptavidin. This signal shows significantoverlap with the WGA signal, indicating the labeled fucosylglycoconjugates are localized in Golgi apparatus (FIGS. 4 and 10).Similar results are obtained from cells treated with alkynyl ManNAc 4,which probes for tagged sialic acid-containing glycoconjugates, whenlabeled by biotin probe 6 and fluorogenic probe 7 (FIGS. 4, 5, 10 and11). Consistent with the results from flow cytometry, confocalmicroscopic analysis of cells treated with control sugars 3 and 5 givesvery low background after reacting with click probes, confirming thelabeling of alkynyl containing glycoconjugates is specific andsensitive.

Labeling of Glycoconjugates in Cell Extract.

Because the herein disclosed labeling system enables the identificationof cellular glycoconjugates, it can also serve well in glyco-proteomicapplications aimed at discovering unknown glycosylated targets fordiagnostic and therapeutic purposes. In one aspect of the disclosure,cell extracts are analyzed after growing cells in a medium containingalkynyl-derivatized sugars to demonstrate the detection of individuallabeled proteins. Soluble lysate fractions are labeled with biotin probe6, fluorogenic probe 7, or a standard azido-derivatized rhodamine probeused in proteomics before separating proteins by SDS/PAGE. As shown inFIG. 6A, specific biotin-labeling signals were detected by Western blotin proteins from cells treated with alkynyl sugars 1 and 4. Positivefluorescent signal was also detected in alkynyl positive protein lysatewhen labeled with fluorogenic 3-azido-7-hydroxycoumarin 7 and rhodamineprobes (FIG. 6B and FIG. 11). Proteins harvested from cells grown withcontrol sugars 3 and 5 and processed utilizing the same cycloadditionlabeling process, showed little to no signal by Western blot orfluorescence. The labeling patterns for Fucyne and ManNAcyne are notablydifferent, indicating the detection of unique glycoconjugates. The dataherein presented demonstrate the feasibility and utility of labeling andidentifying individual glycoconjugates by using this probing system.Moreover, further processing, including a streptavidin/avidin enrichmentor gel slice purification, will allow for comparative identification byproteomic mass spectrometry techniques of unknown glycoconjugatesexpressed at different cell status, for instance, un-differentiatedverses differentiated cells, or normal verses cancer cells.

The ability to visualize and isolate cellular glycoconjugates is usefulto deconvolute the complexity and microheterogeneity that make itdifficult to study their biological function. Toward this goal, severalmetabolic oligosaccharide engineering techniques have been developed,wherein the endogenous biosynthetic machinery for glycosylation isexploited to insert sugar analogs in place of their native counterparts.The tagged glycoconjugates, which contain bioorthogonal chemicalhandles, can then be chemoselectively labeled with a complementaryreactive probe for further manipulation, including visualization orisolation. Recently, we designed a system for incorporating derivatized6-azido derivatized Fuc analogs as cellular glycoconjugate “tags” withsubsequent labeling with alkynyl-derivatized probes using CuAAC. We alsointroduced the use of this process for selective and specific labelingof modified glycoconjugates at the cell surface as well as inintracellular environments. Here, we have expanded the scope of ourspecific glycoconjugate tagging system by establishing that anotheruseful chemical reporter, the alkyne group, can also be used to “tag”cellular glycoconjugates when appended on Fuc and ManNAc derivatives.Similar to its azide counterpart, the alkyne is a small, inert,bioorthogonal group that can be chemoselectively labeled by using clickchemistry. The presently disclosed alkynyl Fuc and ManNAc saccharidesrepresent a robust platform for labeling fucosylated and sialylatedglycoconjugates in vivo. Formerly, azide sugar analogs were incorporatedinto glycoconjugates. However, it has been found that the azido Fucanalog is quite toxic to cells at the levels required for efficientlabeling, which might in turn lead to aberrant cellular glycan profiles.The alkynyl Fuc, on the other hand, is much less toxic, yielding highersignals and less background, when cellular incorporation was monitoredby flow cytometry. Alkynyl-derivatized ManNAc is not toxic at the lowlevels of the modified sugar required for efficient glycoconjugatelabeling as observed by flow cytometry and microscopy. Without beingbound by theory, this likely reflects the higher relative abundance ofsialic acid verses Fuc residues. The alkynyl sugars also are efficientligation partners for click activated fluorogenic and standard clickprobes. Tagging with click-activated probes is particularly usefulbecause of the generation of an instant signal upon ligation withmodified glycoconjugates that does not produce any significantbackground. As established by each of the herein described visualizationmethods (flow cytometry, confocal microscopy, and SDS/PAGE), the signalgenerated by the click-activated probe is equivalent to that of thebiotin-secondary detection systems; however, it requires one lessincubation step and no washing. Furthermore, the click-activated probesare small and hydrophobic, making them more amenable to intracellularpenetration and tagging in living cells. The utility of this approachfor probing interesting glycoconjugates was demonstrated by treatingseveral human cancer cell lines with the alkynyl sugar substrates andsubjecting them to several methods of analysis. In all cases,fluorescent-labeling of cell surface glycoconjugates is witnessed byflow cytometry. Information about intracellular glycoconjugate labelingand localization was determined by using confocal microscopy. Here, itis demonstrated that both alkynyl Fuc and ManNAc modifiedglycoconjugates are localized in the Golgi, consistent with theirproposed site of transfer. Notably, detailed analysis of microscopyimages can supply quantitative data within regions of colocalization,which may provide a useful tool for monitoring glycoconjugate levels andtrafficking.

In another aspect, individual modified glycoconjugates can be separatedand visualized by SDS/PAGE analysis, setting the stage for furtherproteomic analysis. In future studies, we plan to extend and combinethese methodologies to obtain information about cellular glycoconjugatesunder different physiological disease states and cellular statuses, suchas stress, apoptosis, or inflammation. Comparative studies betweenvarious stages of cancer progression, in addition to pulse-chasedtechniques to follow the dynamics of newly synthesized proteins withinindividual cellular systems should provide much needed snapshots ofcritical glycoconjugate behavior. Indeed, in preliminary studies withprostate cancer cells, we observed an increase in the fucosylatedglycoconjugate signal when compared with noncancerous prostate controls(FIG. 10). This indicates that there might be some interestingcorrelations between increased Fuc expression and prostate cancer, afact that is already well known for numerous cancers. Notably, it isimportant to consider that the introduction of modified sugars mightchange the cellular activity of certain glycoconjugates. Perturbationsin glycoconjugate-mediated binding have been noted with viral receptorsand lectin interactions in metabolic oligosaccharide engineering studieswhere sialic acid derivatives were introduced into cellularglycoconjugates. Accordingly, these studies also found that some Fuclectins, including aleuria aurantia lectin (AAL; specific for alpha-1,6-or alpha-1,3-linked Fuc) and Ulex Europaeus Agglutinin I (UEA-1;specific for alpha-1,2-linked Fuc), bound with significantly loweravidity among cells treated with alkyne Fuc verses control (FIG. 11).These results are not surprising, considering that a change from amethyl to a more bulky alkynyl group may interfere with the recognitionin the small conserved hydrophobic pocket found in many Fuc lectins.Indeed, in some cases, altered biological responses may prove useful forperturbing and profiling the function of unknown carbohydrate bindingproteins. On the other hand, some glycoconjugate modifications do notseem to greatly affect binding interactions, past studies analyzing thebinding of selectins to synthetic analogues of sialyl Lewis x showed asignificant tolerance for N-acyl modification on sialic acid. Thus, theanalysis of cells treated with modified sugars over an extended periodmust be evaluated carefully. To circumvent any artifacts from alteredactivity or differential cellular uptake, pulse-chase experiments may beuseful. These experiments would result in lower levels of modifiedglycoconjugates, while presenting a comparable cell-to-cell snap shot ofglycoconjugate behavior.

The usage of alkynyl sugars is further applied to analyze taggedglycoproteomes through metabolic oligosaccharide engineering (MOE) inHelicobacter pylori (H. pylori). Although rare among prokaryotes,Gram-negative bacterium H. pylori possesses the glycosylation machinerynecessary to fucosylate its glycoconjugates. This fucosylation processcan produce Lewis antigens, among other structures, on glycoconjugatesand enables H. pylori to bind to host cells and subsequently evade thehost immune system, thus contributing to persistent infection instomach. MOE strategy provides the opportunity to study fucosylatedglycoconjugates of clinical H. pylori isolates from various stages ofinfection, so that the link between fucosylation and the development ofgastric ulcer and cancer.

Representative H. pylori strains isolated from human gastric biopsyspecimens, including gastritis (HS), duodenal ulcer (HD), gastric ulcer(HU) and gastric cancer (HC), were subjected to MOE: all the strainswere grown on CDC agar plate supplemented with 200 micromolarderivatized alkynyl Fuc 1 for two days under micro-aerobic atmosphere(5% O₂, 15% CO₂, 80% N₂). Tagged protein extracts were prepared in lysisbuffer (1% NP-40, 150 mM NaCl, 100 mM sodium phosphate pH7.5,1×EDTA-free protease inhibitor cocktail) and subjected to subsequentlabeling with biotin probe 6 (protein 1 mg/ml with 0.1 mM azido biotin6/0.1 mM Tris-triazoleamine catalyst/1 mM CuSO₄/2 mM sodium ascorbate inlysis buffer) at room temperature for 1 h. To isolate glycoproteins, 1mg labeled protein samples were precipitated with 10% TCA for 30 mM toremove excessive biotin probe, re-dissolved in 1 ml of 0.2% SDS/PBS, andimmunoprecipitated with 50 μl anti-biotin agarose beads (VectorLaboratories) at room temperature for 1 h Immunoprecipitates were thenanalyzed by SDS-PAGE and stained for visualization. As shown in FIG.13A, several proteins were detected in MOE-tagged H. pylori, while noproteins were isolated from non-tagged H. pylori proteome samples,indicating that the immunoprecipitation process was specific. Notably,more fucosyl proteins were detected in HC and HU strains, and fewerproteins were observed in HS and HD strains. Protein bands revealed inSDS-protein gel (marked with numbers in FIG. 13A) were excised,extracted, reduced, alkylated, tryptic digested to elute peptides andsubjected to LC-MS² analysis for protein identification (FIG. 13B).

To validate the fucosylation of proteins in H. pylori, we examined CagA(cytotoxicity-associated immunodominant antigen), a virulence factorreported to associate with malignancy, for the incorporation of alkynylFuc 1 by anti-CagA antibody in HC strain as follows: Labeled proteinsextracted from control or alkynyl Fuc 1-treated HC samples weresubsequently labeled via-cycloaddition with biotin probe, followed byimmunoprecipitation with anti-CagA antibody. The biotinylated Fuc tagspresent on CagA protein were revealed by peroxidase-conjugatedstreptavidin on protein blot. By comparison with the CagA proteinisolated from a control sample (derived from cells grown without alkynylFuc 1), a specific signal is only detected in MOE-tagged HC strain,indicating the existence of alkynyl Fuc tags on CagA protein (FIG. 14).

Secretory glycoconjugates are known to be continuously recycled.Glycoconjugates are synthesized in the ER/Golgi, and then exported tosubcellular locations, primarily the cell-surface, before beingendocytosed to the lysome, where they are processed and ultimately takenback to the Golgi to start the cycle again. At this point, the kineticsof these processes are not well understood, and conflicting reportsexist. Pulse-chased experiments can be used examine trafficking ofglycans, and to monitor the differential trafficking of glycans in cellsat different stages of disease. It is worth noting, that by pulsing thesugars cellular perturbations caused by the modified architecture of theakynylated glycans and/or toxicity of the azido-fucose derivative may bereduced by use of lower concentrations and time exposure to derivatizedsugars.

Pulse-chased MOE experiments entail growing cells of interest tolog-phase levels and then exposing them to the sugar analogs in thegrowth medium, as prescribed by standard MOE, but only for a 30 minutepulse, before replenishing with fresh medium devoid of sugars. Postsugar-pulse, the cells are grown for various lengths of time beforeanalysis. FIG. 15 shows microscopy of Hep3B cells subjected topulse-chase conditions with Fucyne, CuAAC labeled with biotin, anddetected by streptavidin-FITC. The signal for the fucosylated glycans(green) may emerge as a co-localized yellow signal as early as 2 h,indicating significant overlap with the Golgi marker (red). A pure greensignal may increase over longer periods following the Fucyne pulse,indicating a progression from the golgi to the cell surface (data thatmay be obtained for sialylated alkynyl glycans shows a similar trend).Notably, this progression from Golgi to cell surface using copper freeclick-reactions, label cell-surface glycans.

For example, by coupling the pulse-chase procedure with cellular markersof interest (e.g., to image the lysome with LysoTrackerRed, the Golgiwith BODIPY ceramide TR, the endsome with Alexafluor-labeledtransferrin, or the ER with R6-rhodamine B hexyl-ester chloride)information may be obtained about dynamics and trafficking of glycans incancer cells. Using orthogonal sugar probes, for instance FucAz andManNAcyne (or Fucyne and azido sialic acid derivatives), in combinationwith probe fluorophores that emit at different wavelengths, may allowfor simultaneous labeling and imaging of both sugars in the same sample.A combination of copper-free and CuAAC may also provide more in-depthinformation about cellular trafficking. Pulsing Fucyne and thenazido-derivatized sugars may provide more information about spatial andtemporal trafficking of fucosylated glycoconjugates (First pulse at thecell surface, while second pulse is in ER, etc). Finally, Doublelabeling with FucAz and ManNAcyne may be used to monitor the traffickingof fucosylated verses sialylated glycans, this data may be further usedto quantify, contrast and compare the relative numbers of fucosylatedverses sialylated verses fucosylated and sialylated glycans found at acell in various life cycle stages.

In one exemplary implementation of the disclosure, an Intermediary ofAzido Derivatized Fucose is6,7-Deoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-ynopyranoside Asuspension of PCC (1.3 g, 6.0 mmol), NaOAc (1.0 g, 12.0 mmol) and 4.ANG. molecular sieves (2.7 g) in dry CH₂Cl₂ (18 mL) was stirred for 1h. To this mixture was added a solution of 17 (520 mg, 2.0 mmol) in dryCH₂Cl₂ (9 mL) dropwise, and the mixture was stirred at room temperaturefor 2 h. The reaction mixture was diluted with hexane/ether (1:1, 50mL), and the solution was filtered through a bed of silica gel. Thefiltrate was concentrated to give the crude aldehyde. To a suspension oftBuOK (471 mg, 4.2 mmol) in dry THF (5 mL) was added a solution of(EtO)₂P(O)CHN₂ (748 mg, 4.2 mmol) in THF (5 mL) at −78° C. and themixture was stirred at 5 min under N₂ gas. To this solution, a solutionof the aldehyde in THF (5 mL) was added, and the mixture was allowed towarm to room temperature and continued to stir overnight. The reactionmixture was quenched with 100 mL of water, and the mixture was extractedwith CH₂Cl₂. The extracts were washed with brine, dried over withNa₂SO₄, and evaporated. The residue was purified by flash columnchromatography on silica gel (AcOEt/hexane 1:5) to afford 4 as acolorless oil (295 mg, 62%).

In conclusion, herein disclosed is a mehod for metabolic oligosaccharideengineering that can incorporate alkyne-bearing sugar analogs incellular glycoconjugates. The utility of the alkynyl system has beendemonstrated by incorporating Fuc and ManNAc derivative sugars intocancer cell lines, where they were visualized at the cell surface,intracellularly, and as individual glycoconjugates. The alkynyl Fucsugar was also incorporated into fucoysylated cellular glycans producedby H. pylori, a causative agent of gastric cancer. Sugars were selectedthat report on Fuc (alkynyl Fuc) and sialic acid (alkynyl ManNAc)because these residues, in particular, have been linked to many aberrantglycoconjugates in cancer. Although several glycan epitopes bindingsialic acid and fucose are known, there are likely many other as yetunidentified glycoconjugates and glycan activities that contribute.Identification of these glycan-related biomarkers and targets fortherapeutic intervention is one of the key objectives in our strategy.

EXAMPLES

All chemicals were purchased as reagent grade and used without furtherpurification. Reactions were monitored with analytical thin-layerchromatography (TLC) on silica gel 60 F254 plates and visualized underUV (254 nm) and/or by staining with 5% sulfuric acid or acidic cericammonium molybdate. ¹H- or ¹³C-NMR spectra were measured on a BrukerDRX-500 or DRX-600 using CDCl₃ or DMSO-d₆ as the solvent (1H, 500 or 600MHz; ¹³C, 125 or 150 MHz). Chemical shifts (in ppm) were determinedrelative to either tetramethylsilane (0 ppm) or deuterated chloroform(77 ppm). Mass spectra were obtained by the analytical services of TheScripps Research Institute. Biotin-conjugated Aleuria Aurantia Lectin(AAL), fluorescein-conjugated streptavidin, and fluorescein conjugatedUlex Europaeus Agglutinin I (UEA-1) was purchased from Vectorlaboratories (Burlingame, Calif.). RPMI 1640, DMEM, Alexa Fluor®594-conjugated WGA lectin, and Hoechst 33342 were purchased fromInvitrogen (Carlsbad, Calif.). SuperBlock® Blocking buffer,peroxidase-conjugated goat anti-mouse IgG, and SuperSignal®Chemiluminescent Substrate were obtained from Pierce (Rockford, Ill.).EDTA-free protease inhibitor cocktail and anti-biotin MAb were purchasedfrom Roche Applied Science (Indianapolis, Ind.).

FCS is Fetal Calf Serum. DMEM is Dulbecco's Modified Eagle Medium. RPMI1640 is Roswell Park Memorial Institute Medium 1640. CDC Agar plate isthe CDC formulation of Remel Anaerobic Blood Agar plate, the formulationdeveloped by CDC scientists (Dowell, V. R, and Hawkins, T. M, LaboratoryMethods in Anaerobic Bacteriology, CDC Laboratory Manual, U.S. Dept. ofH.H.S. and CDC, Atlanta, Ga., 1974).

Example 1 Synthesis of 1,2,3,4-tetraacetyl alkynyl fucose (Fuc) (1,mixture of anomers; Scheme 2)

To a flask containing compound 8 (0.05 g, 0.2 mmol), TFA solution (1 ml,90% TFA in H₂O) was slowly added at 0° C. The reaction was stirred onice for 1 h and concentrated in vacuo. The resulting residue was treatedwith pyridine (1 ml), N,Ndimethylaminopyridine (2.0 mg), and aceticanhydride (1 ml), stirred overnight, concentrated, and diluted withdichloromethane. This solution was then sequentially washed with 1 Naqueous HCl, saturated aqueous NaHCO₃, and brine. The organic phase wasdried over anhydrous Na₂CO₃ and concentrated. Silica gel chromatographygave product 1 (0.055 g, 80%, α-pyranoside:β-pyranoside:α-furanoside:β-furanoside=30:51:11:8) as a colorless gum (FIG. 7).Partial ¹H-NMR of mixture (500 MHz, CDCl₃) δ 5.74 (d, J=8.4 Hz, H-1(β-pyr)), 6.24 (s, H-1 (α-fur)), 6.36 (d, J=4.8 Hz, H-1 (β-fur)), 6.43(d, J=2.6 Hz, H-1 (α-pyr)); ESI-TOF-HRMS m/e calculated for (M+Na)⁺C₁₅H₁₈O₉Na 365.0843; found 365.0839.

Example 2 Synthesis of N-4-pentynoylmannosamine (10, Mixture of Anomers;Scheme 3)

A mixture of D-mannosamine hydrochloride (863 mg, 4.0 mmol),N-succinimidyl 4-pentynoate 9 (781 mg, 4.0 mmol), triethylamine (1.67ml, 12.0 mmol) in DMF (31 ml) was stirred at room temperature overnight.The reaction mixture was concentrated in vacuo, and the residue waspurified by flash column chromatography (CHCl₃/MeOH 8:1) to giveN-4-Pentynoylmannosamine, 10 (898 mg, 87%); ¹H-NMR (500 MHz, D₂O) 2.37(t, 2.63H, J=2.5 Hz), 2.48-2.63 (m, J=10.5H), 3.38-3.42 (m, 1H), 3.52(t, 1H, J=10 Hz), 3.63 (t, 1.63H, J=10 Hz), 3.69-3.91 (m, 7.89H), 4.05(dd, 1.63H, J=4.5 and 10 Hz), 4.35 (dd, 1.63H, J=1.5 and 4.5 Hz), 4.47(dd, 1H, J=1.5 and 4.5 Hz), 5.03 (d, 1H, J=1.5 Hz), 5.13 (d, 1.63H,J=1.5 Hz); ¹³C-NMR (125 MHz, D₂O) δ 14.78, 14.91, 34.62, 34.79, 53.67,54.50, 60.91, 60.93, 67.01, 67.28, 69.25, 70.56, 70.71, 72.47, 72.50,76.80, 84.04, 84.45, 93.36, 93.67, 175.68, 176.41; ESI-TOF-HRMS m/ecalculated for (M+H)⁺ C₁₁H₁₇NO₆260.1129; found 260.1120.

Example 3 Synthesis of 1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine(4, Mixture of Anomers; Scheme 3)

A mixture of 10 (123 mg, 0.500 mmol) and acetic anhydride (0.227 ml,2.40 mmol) in pyridine (4 ml) was stirred at room temperature overnight.The reaction mixture was concentrated in vacuo, and the residue wasdissolved in CH₂Cl₂ and washed with water. The organic layer was driedover Na₂SO₄ and evaporated. The residue was purified by flash columnchromatography (AcOEt/Hexane 1:4) to give1,3,4,6-tetra-O-acetyl-N-4-pentynoylmannosamine, 4 (183 mg, 86%); ¹H-NMR(500 MHz, CDCl₃) δ 2.00 (s, 9H), 2.06 (s, 9H), 2.097 (s, 3H), 2.10 (s,3H), 2.11 (s, 3H), 2.14-2.18 (m, 3H), 2.19 (s, 6H), 2.46-2.58 (m, 12H),3.81-3.87 (m, 1H), 4.00-4.15 (m, 5H), 4.23-4.30 (m, 3H), 4.69 (dd, 2H,J=4.5 and 10 Hz), 4.82 (dd, 1H, J=4.5 and 10 Hz), 5.09 (dd, 1H, J=4.5and 10 Hz), 5.17 (t, 1H, J=10 Hz), 5.23 (t, 2H, J=10 Hz), 5.33 (dd, 2H,J=4.5 and 10 Hz), 5.90 (s, 1H), 6.03 (s, 2H), 6.36 (d, 1H, J=9.5 Hz),6.54 (d, 2H, J=9.5 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ 15.29, 15.40, 20.99,21.01, 21.06, 21.09, 21.15, 21.21, 35.51, 35.72, 49.56, 49.80, 62.55,62.70, 65.87, 66.07, 69.25, 70.39, 70.54, 70.63, 71.63, 73.69, 83.07,83.11, 90.98, 92.08, 168.59, 168.81, 170.07, 170.44, 170.51, 170.98,171.82, 172.15; ESI-TOF-HRMS m/e calculated for (M+H)⁺ C₁₉H₂₅NO₁₀428.1551; found 428.1549.

Example 4 Synthesis of 3-azidopropyl biotin amide (6; Scheme 4)

A mixture of D-(+)-biotin (100 mg, 0.41 mmol), 1-azido-3-aminopropane(82 mg, 0.82 mmol) O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (311 mg, 0.82 mmol) and N,N-diisopropylethylamine(106 mg, 0.82 mmol) in DMF (5 ml) was stirred at room temperature for 2h. The reaction mixture was concentrated in vacuo, and the residue waspurified by flash column chromatography (CHCl₃/MeOH 10:1) to give theamide 6 (53 mg, 40%); ¹H-NMR (400 MHz, DMSO-d⁶) δ 1.21-1.35 (m, 4H),1.45-1.55 (m, 3H), 1.60-1.67 (m, 3H), 2.05 (t, 2H, J=7.6 Hz), 2.57 (d,1H, J=12.6 Hz), 2.82 (dd, 1H, J=4.8 and 12.6 Hz), 3.07-3.10 (m, 3H),4.10-4.14 (m, 1H), 4.28-4.32 (m, 1H), 6.36 (s, 1H), 6.42 (s, 1H), 7.84(m, 1H); ESI-TOF-HRMS m/e calculated for (M+H)⁺ C₁₃H₂₃N₆O₂S 327.1598;found 327.1598.

Example 5 Flow Cytometric Analysis of Fluorescent Labeling on PC-3 CellSurface

PC-3 cells were grown in RPMI 1640 (Invitrogen) supplemented with 10%FCS and peracetylated alkynyl Fuc 1 or control Fuc 3 for 3 days at 37°C. Cells were then harvested, washed with 1% FCS/PBS, resuspended (5×105cells) in 100 μl of staining solution (1 μg/ml lectin conjugates in 1%FCS/PBS). Cells were then incubated on ice for 30 min and washed twicewith 1% FCS/PBS. Cells stained with biotin-conjugated AAL weresubsequently stained with fluorescein-conjugated streptavidin (0.5μg/sample in 50 μl of 1% FCS/PBS) for 30 min on ice, and washed threetimes with 1% FCS/PBS. Data were acquired by FACSCalibur, and wereanalyzed by CellQuestPro software (BD Biosciences).

Example 6 Flow Cytometric Analysis of Fluorescent Labeling on JurkatCell Surface

Jurkat cells were cultivated (2×10⁶/10 ml) in RPMI medium 1640supplemented with 10% FCS and various concentrations of peracetylatedalkynyl sugars 1, 2, or 4 or native sugars 3 or 5, for 1-3 days at 37°C. Cells were then harvested, washed with 0.1% FCS/PBS, and resuspended(10⁶ cells) in 100 microliters of click reaction solution (0.1 mM biotinprobe 6 or fluorogenic probe 7/0.1 mM Tris-triazoleamine catalyst/0.1 mMCuSO₄/0.5 mM sodium ascorbate, in PBS). The reaction was incubated atroom temperature for 30 min, and then the cells were washed twice with0.1% FCS/PBS. Cells treated with biotin probe 6 were subsequentlystained with fluorescein-conjugated streptavidin (0.5 microgram persample in 50 microliters of 1% FCS/PBS) for 30 min at 4° C., and washedthree times with 1% FCS/PBS. Data were acquired by BD LSR II withFACSDiva software, and were analyzed by CellQuestPro software (BDBiosciences). Detection of fluorescent adduct with probe 7 was monitoredwith a 408 nm laser and a 440/40 bandpass filter for excitation andemission, respectively.

Example 7 Microscopic Analysis of Fluorescent Labeling in Cells

Human hepatocellular carcinoma cells (Hep3B) or breast adenocarcinomacells (MCF-7) were seeded onto six-well plates (3×10⁵/2 ml per well)containing glass coverslips, and were cultivated in 10% FCS/DMEM or 10%FCS/RPMI medium 1640. Growth medium was supplemented with a controlsugar (200 micromolar Fuc 3 or 25 micromolar ManNAc 5 and analkynyl-modified sugar (alkynyl Fuc 1 or alkynyl ManNAc 4 at the sameconcentration as control sugars). After growing for 3 days, cells oncoverslips were fixed and permeabilized with acetone for 10 min, thensubjected to the probe labeling reaction: 0.1 mM biotin probe 6 orfluorogenic probe 7/0.1 mM Tris-triazoleamine catalyst/1 mM CuSO₄/2 mMsodium ascorbate, in PBS, at room temperature for 30 min. Subsequently,the fixed and labeled cells were rinsed with PBS and stained with AlexaFluor 594-conjugated WGA lectin (2 micrograms/ml in 5% BSA/PBS) and/orfluorescein-conjugated streptavidin (2 micrograms/ml in 5% BSA/PBS) atroom temperature for 30 min. Hoechst 33342 (10 microgram/ml in PBS) wasused to stain nuclei. Fluorescent images were captured by Bio-Rad (CarlZeiss) Radiance 2100 Rainbow laser scanning confocal microscopy system.

Example 8 Labeling and Detection of Glycoconjugates in Cell Extracts

Cells were seeded at 3×10⁶/8 ml per 10-cm dish and treated with controland test sugars (200 micromolar Fuc 3 vs. alkynyl derivatized Fuc 1, or25 micromolar ManNAc 5 vs. alkynyl derivatized ManNAc 2) in growthmedium at 37° C. After 3 days, cell extracts were prepared byresuspending the cells in 1 ml of lysis buffer (1% Nonidet P-40/150 mMNaCl/protease inhibitor/100 mM sodium phosphate, pH 7.5). Proteinextract (1 mg/ml) was labeled for 1 h at room temperature (conditions asoutlined in microscopic analysis; the azido rhodamine probe was a giftfrom Benjamin F Cravatt, The Scripps Research Institute). Labeledprotein lysate was resolved by SDS/PAGE. For immunoblotting ofbiotin-labeled glycoconjugates, electrophoresed proteins weretransferred onto PVDF membranes, blocked for 20 min with SuperBlockBlocking Buffer, probed for 1 h with anti-biotin MAb (1 microgram/ml),and incubated with peroxidase-conjugated goat anti-mouse IgG (1:7, 500dilution) for 30 min. Each step was followed by a wash with 0.02% Tween20/PBS (PBST). Signal was developed with SuperSignal ChemiluminescentSubstrate and detected by exposure to x-ray film. For detecting thecoumarin-labeled glycoconjugates, gels were examined under 365 nm UVlight with a 535+/−50 nm filter. Images were taken by using a BioDoc-Itimaging system (UVP). Rhodamine gels were analyzed.

Example 9 Labeling and Detection of Fucosylated Glycoconjugates in H.pylori

H. pylori isolated from clinical gastric specimens: gastritis (HS),duodenal ulcer (HD), gastric ulcer (HU) and gastric cancer (HC) weregrown on CDC agar plate supplemented with 200 μM derivatized alkynyl Fuc1 for two days under micro-aerobic atmosphere (5% O₂, 15% CO₂, 80% N₂).

Protein extracts were prepared in lysis buffer (1% NP-40, 150 mM NaCl,100 mM sodium phosphate pH 7.5, 1×EDTA-free protease inhibitor cocktail)and labeled with biotin probe 6 (protein 1 mg/ml with 0.1 mM azidobiotin 6/0.1 mM Tris-triazoleamine catalyst/1 mM CuSO4/2 mM sodiumascorbate in lysis buffer) via cycloaddition at room temperature for 1h. Labeled protein samples (1 mg) were then precipitated with 10% TCAfor 30 min to remove excessive biotin probe, re-dissolved in 1 ml of0.2% SDS/PBS, and immunoprecipitated with 50 microliter anti-biotinagarose beads (Vector Laboratories) at room temperature for 1 h.Immunoprecipitates were then analyzed by SDS-PAGE and stained forvisualization. Protein bands were excised from SDS-gel, extracted,reduced, alkylated, and trypsin digested to elute peptides and subjectedto LC-MS/MS analysis for identifying peptide sequences in MS core inGenomics Research Center, Academia Sinica, Taipei, Taiwan.

Detection of CagA on protein blots: Protein extracted from control oralkynyl Fuc 1 analog-treated HC samples were subjected to cycloadditionlabeling to label with biotin probe (protein 1 mg/ml with 0.1 mM azidobiotin 6/0.1 mM Tris-triazoleamine catalyst/1 mM CuSO4/2 mM sodiumascorbate in lysis buffer), followed by immunoprecipitation withanti-CagA antibody (Santa Cruz). The derivatized alkynyl Fuc tags/biotinlabeling on CagA protein were detected by peroxidase-conjugatedstreptavidin on protein blot.

Example 10 Pulse-Chase Analysis of Fucosylated and Sialylated Glycans

Cells of interest may be grown to log phase and then presented withvarious derivatized sugars for a limited period of time; for example, 30minutes. After the limited period of time, the cells are no longerpresented with the various sugars. The cells with glycoconjugatesincorporating various tagged sugars may then be visualized over timethrough secondary detection/and or microscopy in relation to variousintracellular and intercellular structures, in order to collect data onthe trafficking and relative location of tagged and labeledglycoconjugates.

While various exemplary implementations of the present disclosure havebeen described in detail, it is apparent that modifications andadaptations of those exemplary implementations will occur to thoseskilled in the art. However, it is to be expressly understood that suchmodifications and adaptations are within the spirit and scope of thepresent disclosure.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

We claim:
 1. A composition comprising a tagged glycoconjugate whereinthe tagged glycoconjugate is linked to a probe through a triazole moietywherein the tagged glycoconjugate is provided by a process comprising:a) providing the L-enantiomer of the alkynyl derivatized sugar 1 havingthe structure

b) metabolically incorporating the alkynyl derivatized sugar 1 into acellular glycan of a cell via the Fuc salvage pathway thereby producinga tagged glyconjugate in the cell wherein the tagged glycoconjugatecomprises a glycan portion, a conjugate portion from the cellular glycanand the alkyne functional group from the alkyne derivatized sugar, andc) providing a probe having an azide group and linking the probe to thealkyne functional group of the tagged glycoconjugate through a triazolemoiety by an azide-alkyne cycloaddition reaction to provide the taggedglycoconjugate linked to the probe through the triazole moiety.
 2. Thecomposition of claim 1, wherein the probe comprises a directly orindirectly detectable moiety.
 3. The composition of claim 2, wherein thedetectable moiety is selected from the group consisting of: afluorescent moiety, a fluorogenic moiety, a moiety detectable bybiotin-avidin interaction, a moiety detectable by antigen-antibodyinteraction, and a coumarin.
 4. The composition of claim 3, wherein theprobe comprises a fluorogenic detectable moiety which turns fluorescentupon Cu(I) catalyzed [3+2] azide-alkyne cycloaddition with the alkynylfunctional group.
 5. The composition of claim 3, wherein the probecomprises a N-alkyl-1,8-naphthalimide fluorogenic detectable moiety. 6.The composition of claim 1, wherein the resulting toxicity of thecomposition produced in claim 1 is improved by at least 10% as comparedto presenting an azido-derivatized sugar to produce the taggedglycoconjugate.
 7. A compound of the L-enantiomer of thealkynyl-derivatized sugar 1,


8. A kit comprising the compound of claim
 7. 9. A composition comprisingthe L-enantiomer of the alkynyl-derivatized sugar 1,